Three-dimensional pancreatic cell and tissue culture system

ABSTRACT

The present invention relates to a three-dimensional cell culture system which can be used to culture a variety of different cells and tissues in vitro for prolonged periods of time. In accordance with the invention, cells derived from a desired tissue are inoculated and grown on a pre-established stromal support matrix. The stromal support matrix comprises stromal cells, such as fibroblasts actively growing on a three-dimensional matrix. Stromal cells may also include other cells found in loose connective tissue such as endothelial cells, macrophages/monocytes, adipocytes, pericytes, reticular cells found in bone marrow stroma, etc. The stromal matrix provides the support, growth factors, and regulatory factors necessary to sustain long-term active proliferation of cells in culture. When grown in this three-dimensional system, the proliferating cells mature and segregate properly to form components of adult tissues analogous to counterparts found in vivo.

This is a division, of application Ser. No. 08/131,361, filed Oct. 4,1993, U.S. Pat. No. 5,443,950, which is a division of Ser. No.07/575,518, filed Aug. 30, 1990, U.S. Pat. No. 5,266,480; which is adivision of Ser. No. 07/402,104, filed Sep. 1, 1989, U.S. Pat. No.5,032,508; which is a continuation-in-part of Ser. No. 07/242,096, filedSep. 8, 1988, U.S. Pat. No. 4,963,489; which is a continuation-in-partof Ser. No. 07/038,110, filed Apr. 14, 1987, abandoned; which is acontinuation-in-part of Ser. No. 07/036,154, filed Apr. 3, 1987, U.S.Pat. No. 4,721,096 which is a continuation of Ser. No. 06/853,569, filedApr. 18, 1986, abandoned; each of which is incorporated by referenceherein in its entirety.

TABLE OF CONTENTS

1. Introduction

2. Background of the Invention

3. Summary of the Invention

3.1. Definitions and Abbreviations

4. Description of the Figures

5. Detailed Description of the Invention The Three-Dimensional CellCulture System

5.1. Establishment of Three-Dimensional Stromal Matrix

5.2. Inoculation of Tissue-Specific Cells Onto Three-Dimensional StromalMatrix And Maintenance of Cultures

5.3. Uses of the Three-Dimensional Culture System

6. Three-Dimensional Bone Marrow Culture System

6.1. Obtaining Bone Marrow Cells

6.2. Establishment Of The Three-Dimensional Stromal Matrix

6.2.1. Enhancing the Growth of Marrow Stromal Cells

6.3. Inoculation of Hematopoietic Cells

6.4. Long Term Growth of Three-Dimensional Bone Marrow Cultures

6.5. Modulation of Hematopoiesis in Three-Dimensional Long-Term BoneMarrow Culture

6.6. Uses of the Three-Dimensional Bone Marrow Culture System

6.6.1. Transplantation

6.6.2. Monitoring A Patient's Condition.

6.6.3. Screening Compounds

7. Three-Dimensional Skin Culture System

7.1. Establishment of the Three-Dimensional Stromal Support AndFormation of the Dermal Equivalent

7.2. Inoculation of the Dermal Equivalent with Epidermal Cells

7.3. Morphological Characterization of Three-Dimensional Skin Culture

7.4. Transplantation In Vivo

7.5. In Vitro Uses of The Three Dimensional Skin Culture

8. Three-Dimensional Liver Tissue Culture System

9. Three-Dimensional Model System For The Blood Brain Barrier

10. Three-Dimensional Pancreas Tissue Culture System

11. Example: Three-Dimensional Bone Marrow Culture System

11.1. Preparation of Bone Marrow Samples

11.1.1. Human Bone Marrow

11.1.2. Non-Human Primate Bone Marrow

11.1.3. Rat Bone Marrow

11.2. Establishment of the Three-Dimensional Stromal Matrix

11.2.1. Preparation of the Screen and Inoculation of Stromal Cells forHuman LTBMC

11.2.2. Preparation of the Screen and Inoculation of Stromal Cells forNon-Human Primate LTBMC

11.2.3. Preparation of the Screen and Inoculation of Stromal Cells forRat LTBMC

11.3. Inoculation of Three-Dimensional Stromal Matrix with HematopoieticCells and Establishment of Culture

11.4. Evaluation of Three-Dimensional Bone Marrow Culture

11.4.1. Histological Evaluation

11.4.2. Total Cell Counts and Cytospin Analysis of Spent Medium ofThree-Dimensional LTBMC

11.4.3. Total Cell Counts and Cytospin Analysis of Adherent Zone ofThree-Dimensional LTBMC

11.4.4. CFU-C and BFU-E Content of Adherent Zone of Three DimensionalLTBMC

11.4.5. Cytofluorographic Analysis of Cellular Content of Adherent Zoneof Three-Dimensional LTMBC

11.4.6. The Effect of Confluent Stromal Cell Monolayers on Cell Growthin Three-Dimensional Cultures

12. Example: Three-Dimensional Skin Culture System

12.1. Establishment of the Three Dimensional Stroma

12.2. Inoculation of Melanocytes and Keratinocytes

12.3. Histological Analysis of Skin Culture

12.4. Transplantation of Three-Dimensional Skin-Culture In Vivo

13. Example: Three-Dimensional Liver Tissue Culture System

13.1. Materials and Methods

13.1.1. Anesthesia

13.1.2. Dissection

13.1.3. Cell Solution Preparation

13.1.4. Preparation Of Three-Dimensional Stromal Matrix

13.1.5. Maintenance Of Three-Dimensional Liver Tissue Cultures

13.2. Results and Discussion

14. Example: Three-Dimensional Muscosal Tissue Epithelium Culture System

14.1. Materials And Methods

14.1.1. Preparation Of Mucosal Epithelial Cells

14.1.2. Preparation Of The Three-Dimensional Stromal Matrix

14.1.3. Maintenance Of Three-Dimensional Muscosal Epithelium TissueCultures

14.2. Results And Discussion

15. Example: Three-Dimensional Pancreas Tissue Culture System

15.1. Materials And Methods

15.1.1. Preparation Of Pancreatic Acinar Cells

15.1.2. Preparation Of The Three-Dimensional Stromal Matrix

15.1.3. Maintenance Of Three-Dimensional Pancreatic Tissue Cultures

15.2. Results and Discussion

16. Example: Three-Dimensional Model System For The Blood Brain-Barrier

16.1. Materials And Methods

16.1.1. Preparation Of Small Vessel Endothelial Cells

16.1.2. Preparation And Seeding Of Mesh

16.1.3. Preparation Of Neuron And Astrocyte Cell Populations

16.1.4. Seeding The Astrocytes Onto Three-Dimensional Endothelial CellCultures

16.1.5. Seeding Neurons Onto Three-Dimensional EndothelialCell-Astrocyte Tissue Cultures

6.2. Results And Discussion

17. Example: Three-Dimensional Adenocarcinoma Tissue Culture System

17.1 Materials And Methods

17.1.1. Preparation Of Adenocarcinoma Stromal And Parenchymal Cells

17.1.2. Preparation Of The Three-Dimensional Stromal Matrix

17.1.3. Maintenance Of Three-Dimensional Adenocarcinoma Tissue Cultures

7.2. Results And Discussion

18. Example: Three-Dimensional Tissue Culture Cytoxicity Testing System

18.1. Materials And Methods

18.1.1. Preparation Of Three-Dimensional Bone Marrow Tissue Cultures

18.1.2. Exposure Of Three-Dimensional Bone Marrow Cultures To CytotoxicAgents

18.1.3. Cytotoxicity Assay

18.1.4. Solutions For Cytoxicity Assay

18.2. Results And Discussion

19. Example: Three-Dimensional Skin Culture System For ImplantationUsing A Neodermis In Micropigs

19.1. Materials And Methods

19.1.1. Preparation Of The Wound Bed

19.1.2. Anesthesia

19.1.3. Animal Maintenance

19.1.4. Epithelial Grafts

19.2. Results

19.3. Discussion

1. INTRODUCTION

The present invention is directed to a three-dimensional cell and tissueculture system. This culture system can be used for the long termproliferation of cells and tissues in vitro in an environment that moreclosely approximates that found in vivo. The culture system describedherein provides for proliferation and appropriate cell maturation toform structures analogous to tissue counterparts in vivo.

The resulting cultures have a variety of applications ranging fromtransplantation or implantation in vivo, to screening cytotoxiccompounds and pharmaceutical compounds in vitro, and to the productionof biologically active molecules in "bioreactors". The invention isdemonstrated by way of examples describing the three-dimensional cultureof bone marrow, skin, liver, muscosal epithelium, pancreas, andadenocarcinoma, and further examples which show the use ofthree-dimensional culture systems in cytotoxicity assays, a blood-brainbarrier model system and skin transplants.

2. BACKGROUND OF THE INVENTION

The majority of vertebrate cell cultures in vitro are grown asmonolayers on an artificial substrate bathed in nutrient medium. Thenature of the substrate on which the monolayers grow may be solid, suchas plastic, or semisolid gels, such as collagen or agar. Disposableplastics have become the preferred substrate used in modern-day tissueor cell culture.

A few researchers have explored the use of natural substrates related tobasement membrane components. Basement membranes comprise a mixture ofglycoproteins and proteoglycans that surround most cells in vivo. Forexample, Reid and Rojkund (1979, In, Methods in Enzymology, Vol. 57,Cell Culture, Jakoby & Pasten, eds., New York, Acad. Press, pp.263-278):Vlodavsky et al., (1980, Cell 19:607-617); Yang et al., (1979, Proc.Natl. Acad. Sci. USA 76:3401) have used collagen for culturingheptocytes, epithelial cells and endothelial tissue. Growth of cells onfloating collagen (Michalopoulos and Pitot, 1975, Fed. Proc. 34:826) andcellulose nitrate membranes (Savage and Bonney, 1978, Exp. Cell Res.114:307-315) have been used in attempts to promote terminaldifferentiation. However, prolonged cellular regeneration and theculture of such tissues in such systems has not heretofore beenachieved.

Cultures of mouse embryo fibroblasts have been used to enhance growth ofcells, particularly at low densities. This effect is thought to be duepartly to supplementation of the medium but may also be due toconditioning of the substrate by cell products. In these systems, feederlayers of fibroblasts are grown as confluent monolayers which make thesurface suitable for attachment of other cells. For example, the growthof glioma on confluent feeder layers of normal fetal intestine has beenreported (Lindsay, 1979, Nature 228:80).

While the growth of cells in two dimensions is a convenient method forpreparing, observing and studying cells in culture, allowing a high rateof cell proliferation, it lacks the cell-cell and cell-matrixinteractions characteristic of whole tissue in vivo. In order to studysuch functional and morphological interactions, a few investigators haveexplored the use of three-dimensional substrates such as collagen gel(Douglas et al., 1980, In Vitro 16:306-312; Yang et al., 1979, Proc.Natl. Acad. Sci. 76:3401; Yang et al., 1980, Proc. Natl. Acad. Sci.77:2088-2092; Yang et al., 1981, Cancer Res. 41:1021-1027); cellulosesponge, alone (Leighton et al., 1951, J. Natl. Cancer Inst. 12:545-561)or collagen coated (Leighton et al., 1968, Cancer Res. 28:286-296); agelatin sponge, Gelfoam (Sorour et al., 1975, J. Neurosurg. 43:742-749).

In general, these three-dimensional substrates are inoculated with thecells to be cultured. Many of the cell types have been reported topenetrate the matrix and establish a "tissue-like" histology. Forexample, three dimensional collagen gels have been utilized to culturebreast epithelium (Yang et al., 1981, Cancer Res. 41:1021-1027) andsympathetic neurons (Ebendal, 1976, Exp. Cell Res. 98:159-169).Additionally, various attempts have been made to regenerate tissue-likearchitecture from dispersed monolayer cultures. Kruse and Miedema (1965,J. Cell Biol. 27:273) reported that perfused monolayers could grow tomore than ten cells deep and organiod structures can develop inmultilayered cultures if kept supplied with appropriate medium (see alsoSchneider et al., 1963, Exp. Cell Res. 30:449-459 and Bell et al., 1979,Proc. Natl. Acad. Sci. USA 76:1274-1279); Green (1978, Science200:1385-1388) has reported that human epidermal kerotinocytes may formdematoglyphs (friction ridges) if kept for several weeks withouttransfer; Folkman and Haudenschild (1980, Nature 288:551-556) reportedthe formation of capillary tubules in cultures of vascular endothelialcells cultured in the presence of endothelial growth factor and mediumconditioned by tumor cells; and Sirica et al. (1979, Proc. Natl. Acad.Sci. U.S.A. 76:283-287; 1980, Cancer Res. 40:3259-3267) maintainedhepatocytes in primary culture for about 10-13 days on nylon meshescoated with a thin layer of collagen. However, the long term culture andproliferation of cells in such systems has not been achieved.

Indeed, the establishment of long term culture of tissues such as bonemarrow has been attempted. Overall the results were disappointing, inthat although a stromal cell layer containing different cell types israpidly formed, significant hematopoiesis could not be maintained forany real time. (For review see Dexter et al., In Long Term Bone MarrowCulture, 1984, Alan R. Liss, Inc., pp.57-96).

3. SUMMARY OF THE INVENTION

The present invention relates to a three-dimensional cell culture systemwhich can be used to culture a variety of different cells and tissues invitro for prolonged periods of time. In accordance with the invention,cells derived from a desired tissue are inoculated and grown on apre-established stromal support matrix. The stromal support matrixcomprises stromal cells, such as fibroblasts, actively growing on athree-dimensional matrix. Stromal cells may also include other cellsfound in loose connective tissue such as endothelial cells,macrophages/monocytes, adipocytes, pericytes, reticular cells found inbone marrow stroma, etc. The stromal matrix provides the support, growthfactors, and regulatory factors necessary to sustain long-term activeproliferation of cells in culture. When grown in this three-dimensionalsystem, the proliferating cells mature and segregate properly to formcomponents of adult tissues analogous to counterparts found in vivo.

The invention is based, in part, on the discovery that growth of stromalcells in three dimensions will sustain active proliferation of cells inculture for longer periods of time than will monolayer systems. This maybe due, in part, to the increased surface area of the three-dimensionalmatrix which results in a prolonged period of active proliferation ofstromal cells. These proliferating stromal cells elaborate proteins,growth factors and regulatory factors necessary to support the long termproliferation of both stromal and tissue-specific cells inoculated ontothe stromal matrix. In addition, the three-dimensionality of the matrixallows for a spatial distribution which more closely approximatesconditions in vivo, thus allowing for the formation of microenvironmentsconducive to cellular maturation and migration. The growth of cells inthe presence of this support may be further enhanced by adding proteins,glycoproteins, glycosaminoglycans, a cellular matrix, and othermaterials to the support itself or by coating the support with thesematerials.

The use of a three-dimensional support allows the cells to grow inmultiple layers, thus creating the three-dimensional cell culture systemof the present invention. Many cell types and tissues can be grown inthe three-dimensional culture system.

In specific embodiments of the invention, bone marrow, skin, liver,pancreas, mucosal epithelium, adenocarcinoma and melanoma tissues may begrown in the three dimensional culture system.

In addition, the resulting cultures may be used as model systems for thestudy of physiologic or pathologic conditions. For example, in aspecific embodiment of the invention, a three-dimensional culture systemmay be used as a model for the blood-brain barrier. In an additionalspecific embodiment, and not by way of limitation, a three-dimensionalculture of mucosal epithelium may be used as a model system to studyherpesvirus or papillomavirus infection. The resulting cultures have avariety of applications ranging from transplantation or implantation, invivo, of cells grown in the cultures, cytotoxicity testing and screeningcompounds in vitro, and the design of "bioreactors" for the productionof biological materials in vitro.

3.1. DEFINITIONS AND ABBREVIATIONS

The following terms used herein shall have the meanings indicated:

Adherent Layer: cells attached directly to the three-dimensional matrixor connected indirectly by attachment to cells that are themselvesattached directly to the matrix.

Stromal Cells: fibroblasts with or without other cells and/or elementsfound in loose connective tissue, including but not limited to,endothelial cells, pericytes, macrophages, monocytes, plasma cells, mastcells, adipocytes, etc.

Tissue-Specific or Parenchymal Cells: the cells which form the essentialand distinctive tissue of an organ as distinguished from its supportiveframework.

Three-Dimensional Matrix: a three dimensional matrix composed of anymaterial and/or shape that (a) allows cells to attach to it (or can bemodified to allow cells to attach to it); and (b) allows cells to growin more than one layer. This support is inoculated with stromal cells toform the three-dimensional stromal matrix.

Three-Dimensional Stromal Matrix: a three dimensional matrix which hasbeen inoculated with stromal cells. Whether confluent or subconfluent,stromal cells according to the invention continue to grow and divide.The stromal matrix will support the growth of tissue-specific cellslater inoculated to form the three dimensional cell culture.

Three-Dimensional Cell Culture: a three dimensional stromal matrix whichhas been inoculated with tissue-specific cells and cultured. In general,the tissue specific cells used to inoculate the three-dimensionalstromal matrix should include the "stem" cells (or "reserve" cells) forthat tissue; i.e., those cells which generate new cells that will matureinto the specialized cells that form the parenchyma of the tissue.

The following abbreviations shall have the meanings indicated:

BFU-E=burst-forming unit-erythroid

CFU-C=colony forming unit-culture

CFU-GEMM=colony forming unit-granuloid, erythroid, monocyte,megakaryocyte

EDTA=ethylene diamine tetraacetic acid

FBS=fetal bovine serum

HBSS=Hank's balanced salt solution

HS=horse serum

LTBMC=long term bone marrow culture

MEM: minimal essential medium

PBL=peripheral blood leukocytes

PBS=phosphate buffered saline

RPMI 1640=Roswell Park Memorial Institute medium number 1640 (GIBCO,Inc., Grand Island, N.Y.)

SEM=scanning electron microscopy

4. DESCRIPTION OF THE FIGURES

FIG. 1 is a scanning electron micrograph depicting fibroblast attachmentto the three-dimensional matrix and extension of cellular processesacross the mesh opening. Fibroblasts are actively secreting matrixproteins and are at the appropriate stage of subconfluency which shouldbe obtained prior to inoculation with tissue-specific cells.

FIG. 2 is a scanning electron micrograph of the three-dimensional LTBMCdemonstrating the 210 μm sieve area for expression of erythroid, myeloidand other colonies. Support cells have grown linearly along andenveloped the three-dimensional matrix.

FIG. 3 is a graph representing the total cell count of thethree-dimensional LTBMC adherent and nonadherent layers over severalweeks in culture. Total cell counts and cytospin preparations of thenonadherent zone were made using spent medium removed when the cultureswere fed every five days. Cell counts of the adherent zone were done atdifferent intervals of LTBMC by treating the three-dimensional cellculture with collagenase and trypsin to remove adherent cells. Cellularproliferation achieved a steady state condition after several weeks inculture.

FIG. 4 is a graph representing the CFU-C per 10⁵ cells obtained from theadherent zone of the three-dimensional LTBMC over several weeks inculture.

FIG. 5 is a diagrammatic representation of the three-dimensional skinmodel. A dermal/epidermal junction is present, above which liespigmented melanocytes and several layers of pigment-containingkeratinocytes. The stromal cells attach to the matrix and form thedermal component.

FIG. 6 is a scanning electron micrograph of the three-dimensional stromathree days after inoculation with melanocytes. Melanocytes grow normallyin the three-dimensional system in that they exhibit dendrite formation,remain pigmented, and retain the ability to transfer pigment tokeratinocytes.

FIG. 7 is a photomicrograph of a cross section of the three-dimensionalskin culture stained with hematoxylineosin. Normal epidermal (E) cellmorphology and orientation is obvious. Epidermal and dermal (D)components completely surround the mesh fiber (M), and a distinctdermal/epidermal junction is present.

FIG. 8 is a photomicrograph showing an area of epidermis from thethree-dimensional skin culture stained with toluidine. Keratinocytes (K)manifest a normal morphology and contain pigment (P) granules. Amaturation of cells is seen, with evidence of stratum corenum (SC).

FIG. 9 is a photomicrograph of the three-dimensional skin model graftedonto rats seven days post transplant. A distinct dermal and epidermaljunction is evident. Cells show firm attachment to the mesh with nosigns of rejection.

FIG. 10 is a photomicrograph of the three-dimensional skin model graftedonto rats seven days post transplant. Collagen bundles (c) and all celltypes are represented, including keratinocytes (k), fibroblasts (f),adipocytes (a), and smooth muscle cells (s), arranged in a naturalconfiguration around the nylon mesh fiber (m).

FIG. 11. is a photomicrograph of adult liver cultures grown by thethree-dimensional culture method forming a three-dimensionalmultilayered tissue on hepatic stromal cells.

FIG. 12. is a photomicrograph of actively dividing hepatocytes duringthe first ten to twelve days after inoculation into three-dimensionalcultures resemble hepatoblasts or cells of regenerating liver.

FIG. 13. is a photomicrograph of a cross-section of a three-dimensionaltissue culture of mucosal epithelium.

FIG. 14. is a photomicrograph of a cross-section of a three-dimensionaltissue culture of pancreas. An arrow points to zymogen granules in anacinar cell. An asterisk indicates a stromal cell.

FIG. 15. is a photomicrograph of a cross-section of a three-dimensionaltissue culture model system of the blood brain barrier. A closed arrowpoints to a small blood vessel endothelial cell. An open arrow points toa neuronal cell. An asterisk indicates an astrocyte.

FIG. 16. is a photomicrograph of a cross-section of a three-dimensionaltissue culture of adenocarcinoma.

FIG. 17. is a graph comparing the response of fibroblasts grown inmonolayer with stromal and full-thickness marrow grown on thethree-dimensional mesh system of the invention. The substrates show adose-related response to adriamycin utilizing the neutral-red assay forcell viability.

FIG. 18. is a graph presenting neutral red assay results showing adose-related response to cis-platinum by stromal and bone marrowthree-dimensional cultures.

FIG. 19. is a photograph showing the surface condition of afull-thickness wound 10 days after implantation of a human neodermisinto micropig. Minimal contraction was noted, with no signs of rejectionor dehydration.

FIG. 20. is a photomicrograph presenting histological evaluation of aneodermis showing a cross-section of mesh fibers, along with activefibroblasts and naturally-secreted collagen.

FIG. 21. is a photograph comparing wounds treated either with neodermis(left) and biodegradable mesh alone (right). Note the decrease incontraction and increase in pigmentation and hair growth in the woundinto which the neodermis was implanted.

FIG. 22. is a photomicrograph showing histological evaluation of abiopsy taken from site treated with mesh soaked in human dermalfibroblast lysate. Note the increase in epithelial cell migration aroundindividual mesh fibers.

FIG. 23. is a photomicrograph showing histological evaluation of adermal equivalent 21 days after implantation. Epithelial cells havemigrated onto the dermal surface, attached evenly, and exhibit normaldifferentiation and growth. The growth of deep rete pegs ischaracteristic of transplanted skin. Resolution of the rete pegs is seenwithin three to four months.

FIG. 24. is a photograph of a full thickness wound 21 days aftertreatment with neodermis. Half of the neodermis has received anautologous cultured epithelial graft. The epithelial graft healedevenly, prevented further contraction, and firmly attached to theunderlying dermal equivalent.

FIG. 25. is a photomicrograph showing histological evaluation of theepidermal/dermal site depicted in FIG. N. Note the even growth andattachment of the keratinocytes to the dermal equivalent. Mesh fibersare still evident 21 days after transplant and fibroblasts remain activeamong naturally secreted collagen fibers.

5. DETAILED DESCRIPTION OF THE INVENTION THE THREE-DIMENSIONAL CELLCULTURE SYSTEM

The present invention involves a three-dimensional matrix and its use asthe framework for a three-dimensional, multi-layer cell culture system.In previously known tissue culture systems, the cells were grown in amonolayer. Cells grown on a three-dimensional stromal support, inaccordance with the present invention, grow in multiple layers, forminga cellular matrix. This matrix system approaches physiologic conditionsfound in vivo to a greater degree than previously described monolayertissue culture systems. The three-dimensional cell culture system isapplicable to the proliferation of different types of cells andformation of a number of different tissues, including but not limited tobone marrow, skin, liver, pancreas, kidney, adrenal and neurologictissue, to name but a few.

The culture system has a variety of applications. For example, fortissues such as skin, glands, etc. the three-dimensional culture itselfmay be transplanted or implanted into a living organism. Alternatively,for diffuse tissues such as bone marrow, the proliferating cells couldbe isolated from the culture system for transplantation. Thethree-dimensional cultures may also be used in vitro for cytotoxicitytesting and screening compounds. In yet another application, thethree-dimensional culture system may be used as a "bioreactor" toproduce cellular products in quantity.

In accordance with the invention, cells derived from a desired tissue(herein referred to as tissue-specific cells or parenchymal cells) areinoculated and cultured on a pre-established three-dimensional stromalmatrix. The stromal matrix comprises stromal cells grown on athree-dimensional matrix or network. The stromal cells comprisefibroblasts with or without additional cells and/or elements describedmore fully herein. The fibroblasts and other cells and/or elements thatcomprise the stroma may be fetal or adult in origin, and may be derivedfrom convenient sources such as skin, liver, pancreas, etc. Such tissuesand/or organs can be obtained by appropriate biopsy or upon autopsy. Infact, cadaver organs may be used to provide a generous supply of stromalcells and elements.

Fetal fibroblasts will support the growth of many different cells andtissues in the three-dimensional culture system, and, therefore, can beinoculated onto the matrix to form a "generic" stromal support matrixfor culturing any of a variety of cells and tissues. However, in certaininstances, it may be preferable to use a "specific" rather than"generic" stromal support matrix, in which case stromal cells andelements can be obtained from a particular tissue, organ, or individual.For example, where the three-dimensional culture is to be used forpurposes of transplantation or implantation in vivo, it may bepreferable to obtain the stromal cells and elements from the individualwho is to receive the transplant or implant. This approach might beespecially advantageous where immunological rejection of the transplantand/or graft versus host disease is likely. Moreover, fibroblasts andother stromal cells and/or elements may be derived from the same type oftissue to be cultured in the three-dimensional system. This might beadvantageous when culturing tissues in which specialized stromal cellsmay play particular structural/functional roles; e.g., glial cells ofneurological tissue, Kupffer cells of liver, etc.

Once inoculated onto the three-dimensional matrix, the stromal cellswill proliferate on the matrix and support the growth of tissue-specificcells inoculated into the three-dimensional culture system of theinvention. In fact, when inoculated with the tissue-specific cells, thethree-dimensional stromal support matrix will sustain activeproliferation of the culture for long periods of time. Growth andregulatory factors may be added to the culture, but are not necessarysince they are elaborated by the stromal support matrix.

Because, according to the invention, it is important to recreate, inculture, the cellular microenvironment found in vivo for a particulartissue, the extent to which the stromal cells are grown prior toinoculation of parenchymal cells may vary depending on the type oftissue to be grown in three-dimensional tissue culture. For example, inbone marrow three-dimensional cultures, it is preferable to inoculatehematopoietic cells onto a stromal matrix which is subconfluent.However, in skin three-dimensional tissue cultures, it is preferred,according to the invention, to allow the stromal cells to reachconfluence prior to inoculation with keratinocytes and/or melanocytes,so as to recreate the structure of the dermal component of skin.Importantly, because openings in the mesh permit the exit of stromalcells in culture, confluent stromal cultures do not exhibit contactinhibition, and the stromal cells continue to grow, divide, and remainfunctionally active.

The invention is based, in part, upon the discovery that growth of thestromal cells in three dimensions will sustain active proliferation ofboth the stromal and tissue-specific cells in culture for much longertime periods than will monolayer systems. Moreover, thethree-dimensional system supports the maturation, differentiation, andsegregation of cells in culture in vitro to form components of adulttissues analogous to counterparts found in vivo.

Although the applicants are under no duty or obligation to explain themechanism by which the invention works, a number of factors inherent inthe three-dimensional culture system may contribute to its success:

(a) The three-dimensional matrix provides a greater surface area forprotein attachment, and consequently, for the adherence of stromalcells.

(b) Because of the three-dimensionality of the matrix, stromal cellscontinue to actively grow, in contrast to cells in monolayer cultures,which grow to confluence, exhibit contact inhibition, and cease to growand divide. The elaboration of growth and regulatory factors byreplicating stromal cells may be partially responsible for stimulatingproliferation and regulating differentiation of cells in culture.

(c) The three-dimensional matrix allows for a spatial distribution ofcellular elements which is more analogous to that found in thecounterpart tissue in vivo.

(d) The increase in potential volume for cell growth in thethree-dimensional system may allow the establishment of localizedmicroenvironments conducive to cellular maturation.

(e) The three-dimensional matrix maximizes cell-cell interactions byallowing greater potential for movement of migratory cells, such asmacrophages, monocytes and possibly lymphocytes in the adherent layer.

(f) It has been recognized that maintenance of a differentiated cellularphenotype requires not only growth/differentiation factors but also theappropriate cellular interactions. The present invention effectivelyrecreates the tissue microenvironment.

The three-dimensional stromal support, the culture system itself, andits maintenance, as well as various uses of the three-dimensionalcultures are described in greater detail in the subsections below.

5.1. ESTABLISHMENT OF THREE-DIMENSIONAL STROMAL MATRIX

The three-dimensional support may be of any material and/or shape that:(a) allows cells to attach to it (or can be modified to allow cells toattach to it); and (b) allows cells to grow in more than one layer. Anumber of different materials may be used to form the matrix, includingbut not limited to: nylon (polyamides), dacron (polyesters),polystyrene, polypropylene, polyacrylates, polyvinyl compounds (e.g.,polyvinylchloride), polycarbonate (PVC), polytetrafluorethylene (PTFE;teflon), thermanox (TPX), nitrocellulose, cotton, polyglycolic acid(PGA), cat gut sutures, cellulose, gelatin, dextran, etc. Any of thesematerials may be woven into a mesh, for example, to form thethree-dimensional matrix. Certain materials, such as nylon, polystyrene,etc., are poor substrates for cellular attachment. When these materialsare used as the three-dimensional support matrix, it is advisable topre-treat the matrix prior to inoculation of stromal cells in order toenhance the attachment of stromal cells to the matrix. For example,prior to inoculation with stromal cells, nylon matrices could be treatedwith 0.1M acetic acid, and incubated in polylysine, FBS, and/or collagento coat the nylon. Polystyrene could be similarly treated using sulfuricacid.

Where the three-dimensional culture is itself to be implanted in vivo,it may be preferable to use biodegradable matrices such as polyglycolicacid, catgut suture material, or gelatin, for example. Where thecultures are to be maintained for long periods of time or cryopreserved,non-degradable materials such as nylon, dacron, polystyrene,polyacrylates, polyvinyls, teflons, cotton, etc. may be preferred. Aconvenient nylon mesh which could be used in accordance with theinvention is Nitex, a nylon filtration mesh having an average pore sizeof 210 μm and an average nylon fiber diameter of 90 μm (#3-210/36,Tetko, Inc., New York).

Stromal cells comprising fibroblasts, with or without other cells andelements described below, are inoculated onto the matrix. Thesefibroblasts may be derived from organs, such as skin, liver, pancreas,etc. which can be obtained by biopsy (where appropriate) or uponautopsy. In fact fibroblasts can be obtained in quantity ratherconveniently from any appropriate cadaver organ. As previouslyexplained, fetal fibroblasts can be used to form a "generic"three-dimensional stromal matrix that will support the growth of avariety of different cells and/or tissues. However, a "specific" stromalmatrix may be prepared by inoculating the three-dimensional matrix withfibroblasts derived from the same type of tissue to be cultured and/orfrom a particular individual who is later to receive the cells and/ortissues grown in culture in accordance with the three-dimensional systemof the invention.

Fibroblasts may be readily isolated by disaggregating an appropriateorgan or tissue which is to serve as the source of the fibroblasts. Thismay be readily accomplished using techniques known to those skilled inthe art. For example, the tissue or organ can be disaggregatedmechanically and/or treated with digestive enzymes and/or chelatingagents that weaken the connections between neighboring cells making itpossible to disperse the tissue into a suspension of individual cellswithout appreciable cell breakage. Enzymatic dissociation can beaccomplished by mincing the tissue and treating the minced tissue withany of a number of digestive enzymes either alone or in combination.These include but are not limited to trypsin, chymotrypsin, collagenase,elastase, and/or hyaluronidase, DNase, pronase, dispase etc. Mechanicaldisruption can also be accomplished by a number of methods including,but not can then be grown to confluency, lifted from the confluentculture and inoculated onto the three-dimensional matrix (see, Naughtonet al., 1987, J. Med. 18(3&4):219-250). Inoculation of thethree-dimensional matrix with a high concentration of stromal cells,e.g., approximately 10⁶ to 5×10⁷ cells/ml, will result in theestablishment of the three-dimensional stromal support in shorterperiods of time.

In addition to fibroblasts, other cells may be added to form thethree-dimensional stromal matrix required to support long term growth inculture. For example, other cells found in loose connective tissue maybe inoculated onto the three-dimensional support along with fibroblasts.Such cells include but are not limited to endothelial cells, pericytes,macrophages, monocytes, plasma cells, mast cells, adipocytes, etc. Thesestromal cells may readily be derived from appropriate organs such asskin, liver, etc., using methods known in the art such as thosediscussed above. In one embodiment of the invention, stromal cells whichare specialized for the particular tissue to be cultured may be added tothe fibroblast stroma. For example, stromal cells of hematopoietictissue, including but not limited to fibroblasts, endothelial cells,macrophages/monocytes, adipocytes and reticular cells, could be used toform the three-dimensional subconfluent stroma for the long term cultureof bone marrow in vitro. Hematopoietic stromal cells may be readilyobtained from the "buffy coat" formed in bone marrow suspensions bycentrifugation at low forces, e,g., 3000×g. Stromal cells of liver mayinclude fibroblasts, Kupffer cells, and vascular and bile ductendothelial cells. Similarly, glial cells could be used as the stroma tosupport the proliferation of neurological cells and tissues; glial cellsfor this purpose can be obtained by trypsinization or collagenasedigestion of embryonic or adult brain (Ponten and Westermark, 1980, inlimited to the use of grinders, blenders, sieves, homogenizers, pressurecells, or insonators to name but a few. For a review of tissuedisaggregation techniques, see Freshney, Culture of Animal Cells. AManual of Basic Technique, 2d Ed., A. R. Liss, Inc., New York, 1987, Ch.9, pp. 107-126.

Once the tissue has been reduced to a suspension of individual cells,the suspension can be fractionated into subpopulations from which thefibroblasts and/or other stromal cells and/or elements can be obtained.This also may be accomplished using standard techniques for cellseparation including but not limited to cloning and selection ofspecific cell types, selective destruction of unwanted cells (negativeselection), separation based upon differential cell agglutinability inthe mixed population, freeze-thaw procedures, differential adherenceproperties of the cells in the mixed population, filtration,conventional and zonal centrifugation, centrifugal elutriation(counterstreaming centrifugation), unit gravity separation,countercurrent distribution, electrophoresis and fluorescence-activatedcell sorting. For a review of clonal selection and cell separationtechniques, see Freshney, Culture of Animal Cells. A Manual of BasicTechniques, 2d Ed., A. R. Liss, Inc., New York, 1987, Ch. 11 and 12, pp.137-168.

The isolation of fibroblasts may, for example, be carried out asfollows: fresh tissue samples are thoroughly washed and minced in Hanksbalanced salt solution (HBSS) in order to remove serum. The mincedtissue is incubated from 1-12 hours in a freshly prepared solution of adissociating enzyme such as trypsin. After such incubation, thedissociated cells are suspended, pelleted by centrifugation and platedonto culture dishes. All fibroblasts will attach before other cells,therefore, appropriate stromal cells can be selectively isolated andgrown. The isolated fibroblasts Federof, S. Hertz, L., eds, "Advances inCellular Neurobiology," Vol.1, New York, Academic Press, pp.209-227).

Again, where the cultured cells are to be used for transplantation orimplantation in vivo it is preferable to obtain the stromal cells fromthe patient's own tissues. The growth of cells in the presence of thethree-dimensional stromal support matrix may be further enhanced byadding to the matrix, or coating the matrix support with proteins (e.g.,collagens, elastic fibers, reticular fibers) glycoproteins,glycosaminoglycans (e.g., heparan sulfate, chondroitin-4-sulfate,chondroitin-6-sulfate, dermatan sulfate, keratan sulfate, etc.), acellular matrix, and/or other materials.

After inoculation of the stromal cells, the three-dimensional matrixshould be incubated in an appropriate nutrient medium. Many commerciallyavailable media such as RPMI 1640, Fisher's, Iscove's, McCoy's, and thelike may be suitable for use. It is important that the three-dimensionalstromal matrix be suspended or floated in the medium during theincubation period in order to maximize proliferative activity. Inaddition, the culture should be "fed" periodically to remove the spentmedia, depopulate released cells, and add fresh media.

During the incubation period, the stromal cells will grow linearly alongand envelop the three-dimensional matrix before beginning to grow intothe openings of the matrix. It is important to grow the cells to anappropriate degree which reflects the amount of stromal cells present inthe in vivo tissue prior to inoculation of the stromal matrix with thetissue-specific cells.

The openings of the matrix should be of an appropriate size to allow thestromal cells to stretch across the openings. Maintaining activelygrowing stromal cells which stretch across the matrix enhances theproduction of growth factors which are elaborated by the stromal cells,and hence will support long term cultures. For example, if the openingsare too small, the stromal cells may rapidly achieve confluence but beunable to easily exit from the mesh; trapped cells may exhibit contactinhibition and cease production of the appropriate factors necessary tosupport proliferation and maintain long term cultures. If the openingsare too large, the stromal cells may be unable to stretch across theopening; this will also decrease stromal cell production of theappropriate factors necessary to support proliferation and maintain longterm cultures. When using a mesh type of matrix, as exemplified herein,we have found that openings ranging from about 150 μm to about 220 μmwill work satisfactorily. However, depending upon the three-dimensionalstructure and intricacy of the matrix, other sizes may work equallywell. In fact, any shape or structure that allow the stromal cells tostretch and continue to replicate and grow for lengthy time periods willwork in accordance with the invention.

Different proportions of the various types of collagen deposited on thematrix can affect the growth of the later inoculated tissue-specificcells. For example, for optimal growth of hematopoietic cells, thematrix should preferably contain collagen types III, IV and I in anapproximate ratio of 6:3:1 in the initial matrix. For three-dimensionalskin culture systems, collagen types I and III are preferably depositedin the initial matrix. The proportions of collagen types deposited canbe manipulated or enhanced by selecting fibroblasts which elaborate theappropriate collagen type. This can be accomplished using monoclonalantibodies of an appropriate isotype or subclass that is capable ofactivating complement, and which define particular collagen types. Theseantibodies and complement can be used to negatively select thefibroblasts which express the desired collagen type. Alternatively, thestroma used to inoculate the matrix can be a mixture of cells whichsynthesize the appropriate collagen types desired. The distribution andorigins of the five types of collagen is shown in Table I.

                  TABLE I                                                         ______________________________________                                        DISTRIBUTIONS AND                                                             ORIGINS OF THE FIVE TYPES OF COLLAGEN                                         Collagen Principal                                                            Type     Tissue Distribution                                                                              Cells of Origin                                   ______________________________________                                        I        Loose and dense ordinary                                                                         Fibroblasts and                                            connective tissue; collagen                                                                      reticular cells;                                           fibers             smooth muscle                                              Fibrocartilage     cells                                                      Bone               Osteoblast                                                 Dentin             Odontoblasts                                      II       Hyaline and elastic cartilage                                                                    Chondrocytes                                               Vitreous body of eye                                                                             Retinal cells                                     III      Loose connective tissue;                                                                         Fibroblasts and                                            reticular fibers   reticular cells                                            Papillary layer of dermis                                                                        Smooth muscle                                              Blood vessels      cells; endo-                                                                  thelial cells                                     IV       Basement membranes Epithelial and                                                                endothelial                                                                   cells                                                      Lens capsule of eye                                                                              Lens fibers                                       V        Fetal membranes; placenta                                                                        Fibroblasts                                                Basement membranes                                                            Bone                                                                          Smooth muscle      Smooth muscle                                                                 cells                                             ______________________________________                                    

Thus, depending upon the tissue to be cultured and the collagen typesdesired, the appropriate stromal cell(s) may be selected to inoculatethe three-dimensional matrix.

During incubation of the three-dimensional stromal support,proliferating cells may be released from the matrix. These releasedcells may stick to the walls of the culture vessel where they maycontinue to proliferate and form a confluent monolayer. This should beprevented or minimized, for example, by removal of the released cellsduring feeding, or by transferring the three-dimensional stromal matrixto a new culture vessel. The presence of a confluent monolayer in thevessel will "shut down" the growth of cells in the three-dimensionalmatrix and/or culture. Removal of the confluent monolayer or transfer ofthe matrix to fresh media in a new vessel will restore proliferativeactivity of the three-dimensional culture system. Such removal ortransfers should be done in any culture vessel which has a stromalmonolayer exceeding 25% confluency. Alternatively, the culture systemcould be agitated to prevent the released cells from sticking, orinstead of periodically feeding the cultures, the culture system couldbe set up so that fresh media continuously flows through the system. Theflow rate could be adjusted to both maximize proliferation within thethree-dimensional culture, and to wash out and remove cells releasedfrom the matrix, so that they will not stick to the walls of the vesseland grow to confluence. In any case, the released stromal cells can becollected and cryopreserved for future use.

5.2. INOCULATION OF TISSUE-SPECIFIC CELLS ONTO THREE-DIMENSIONAL STROMALMATRIX AND MAINTENANCE OF CULTURES

Once the three-dimensional stromal matrix has reached the appropriatedegree of growth, the tissue-specific cells (parenchymal cells) whichare desired to be cultured are inoculated onto the stromal matrix. Ahigh concentration of cells in the inoculum will advantageously resultin increased proliferation in culture much sooner than will lowconcentrations. The cells chosen for inoculation will depend upon thetissue to be cultured, which may include but is not limited to bonemarrow, skin, liver, pancreas, kidney, neurological tissue, and adrenalgland, to name but a few.

For example, and not by way of limitation, a variety of epithelial cellscan be cultured on the three-dimensional living stromal support.Examples of such epithelial cells include, but are not limited to, oralmucosa and gastrointestional (G.I.) tract cells. Such epithelial cellsmay be isolated by enzymatic treatment of the tissue according tomethods known in the art, followed by expansion of these cells inculture and application of epithelial cells to the three-dimensionalstromal support cell matrix (neo-submucosa). The presence of thesubmucosa provides growth factors and other proteins which promotenormal division and differentiation of the oral mucosa cells and thecells ok the G.I. tract lining. Using this methodology other epithelialcells can be grown successfully, including nasal epithelium, respiratorytract epithelium, vaginal epithelium, and corneal epithelium.

A variety of tumors may be grown on the three-dimensional living stromalsupport. Examples of such tumors include but are not limited toadenocarcinoma and malignant melanoma which may be derived from primaryor metastatic sites. Such cultures may be established in a mannersimilar to other three-dimensional epithelial cultures. Briefly, stromalcells, derived from either the patient's tumor or normal tissue or froman allogeneic source, are established on the mesh. After reachingnear-confluency the stromal cells are inoculated with tumor cells. Thetumor cells will continue to divide rapidly and form a three-dimensionalsolid tumor. Tumor cells grown in such a three-dimensional supportexhibit a morphology similar to the in vivo state and express and shedsurface antigens in a manner similar to that of solid tumors; malignantcells grown in monolayers do not exhibit the same degree of similarityto in vivo tumor tissue. Such a physiological growth of tumor cellsallows applications in the study and development of new chemotherapeuticagents, individualized chemotherapy regimens, and mechanisms ofmetastasis. In addition such tumor cultures may be useful inindividualized immunotherapy. In this regard experimentation with ⁵¹ CRrelease studies has indicated that Lak cells evoke a much more potentresponse against tumor cells grown in three-dimensions as compared tocells cultured in monolayer. Immune cells may be obtained from patientsby traditional pheresis techiques and sensitized to the patient's owntumor cells grown in three-dimensional culture.

In general, this inoculum should include the "stem" cell (also calledthe "reserve" cell) for that tissue; i.e., those cells which generatenew cells that will mature into the specialized cells that form thevarious components of the tissue.

The parenchymal or tissue-specific cells used in the inoculum may beobtained from cell suspensions prepared by disaggregating the desiredtissue using standard techniques described for obtaining stromal cellsin Section 5.1 above. The entire cellular suspension itself could beused to inoculate the three-dimensional stromal support matrix. As aresult, the regenerative cells contained within the homogenate willproliferate, mature, and differentiate properly on the matrix, whereasnon-regenerative cells will not. Alternatively, particular cell typesmay be isolated from appropriate fractions of the cellular suspensionusing standard techniques described for fractionating stromal cells inSection 5.1 above. Where the "stem" cells or "reserve" cells can bereadily isolated, these may be used to preferentially inoculate thethree-dimensional stromal support. For example, when culturing bonemarrow, the three-dimensional stroma may be inoculated with bone marrowcells, either fresh or derived from a cryopreserved sample. Whenculturing skin, the three-dimensional stroma may be inoculated withmelanocytes and keratinocytes. When culturing liver, thethree-dimensional stroma may be inoculated with hepatocytes. Whenculturing pancreas, the three-dimensional stroma may be inoculated withpancreatic endocrine cells. For a review of methods which may beutilized to obtain parenchymal cells from various tissues, see,Freshney, Culture of Animal Cells. A Manual of Basic Technique, 2d Ed.,A. R. Liss, Inc., New York, 1987, Ch. 20, pp. 257-288.

During incubation, the three-dimensional cell culture system should besuspended or floated in the nutrient medium. Cultures should be fed withfresh media periodically. Again, care should be taken to prevent cellsreleased from the culture from sticking to the walls of the vessel wherethey could proliferate and form a confluent monolayer. The release ofcells from the three-dimensional culture appears to occur more readilywhen culturing diffuse tissues as opposed to structured tissues. Forexample, the three-dimensional skin culture of the invention ishistologically and morphologically normal; the distinct dermal andepidermal layers do not release cells into the surrounding media. Bycontrast, the three-dimensional bone marrow cultures of the inventionrelease mature non-adherent cells into the medium much the way suchcells are released in marrow in vivo. As previously explained, shouldthe released cells stick to the culture vessel and form a confluentmonolayer, the proliferation of the three-dimensional culture will be"shut down". This can be avoided by removal of released cells duringfeeding, transfer of the three-dimensional culture to a new vessel, byagitation of the culture to prevent sticking of released cells to thevessel wall, or by the continuous flow of fresh media at a ratesufficient to replenish nutrients in the culture and remove releasedcells. In any case, the mature released cells could be collected andcryopreserved for future use.

Growth factors and regulatory factors need not be added to the mediasince these types of factors are elaborated by the three-dimensionalstromal cells. However, the addition of such factors, or the inoculationof other specialized cells may be used to enhance, alter or modulateproliferation and cell maturation in the cultures. The growth andactivity of cells in culture can be affected by a variety of growthfactors such as insulin, growth hormone, somatomedins, colonystimulating factors, erythropoietin, epidermal growth factor, hepaticerythropoietic factor (hepatopoietin), and liver-cell growth factor.Other factors which regulate proliferation and/or differentiationinclude prostaglandins, interleukins, and naturally-occurring chalones.

5.3. USES OF THE THREE-DIMENSIONAL CULTURE SYSTEM

The three-dimensional culture system of the invention can be used in avariety of applications. These include but are not limited totransplantation or implantation of either the cultured cells obtainedfrom the matrix, or the cultured matrix itself in vivo; screeningcytotoxic compounds, allergens, growth/regulatory factors,pharmaceutical compounds, etc., in vitro; elucidating the mechanism ofcertain diseases; studying the mechanism by which drugs and/or growthfactors operate; diagnosing and monitoring cancer in a patient; genetherapy; and the production of biologically active products, to name buta few.

For transplantation or implantation in vivo, either the cells obtainedfrom the culture or the entire three-dimensional culture could beimplanted, depending upon the type of tissue involved. For example,three-dimensional bone marrow cultures can be maintained in vitro forlong periods; the cells isolated from these cultures can be used intransplantation or the entire culture may be implanted. By contrast, inskin cultures, the entire three-dimensional culture can be grafted invivo for treating burn victims, skin ulcerations, wounds, etc.

Three-dimensional tissue culture implants may, according to theinvention, be used to replace or augment existing tissue, to introducenew or altered tissue, to modify artificial prostheses, or to jointogether biological tissues or structures. For example, and not by wayof limitation, specific embodiments of the invention would include (i)three-dimensional bone marrow culture implants used to replace bonemarrow destroyed during chemotherapeutic treatment; (ii)three-dimensional liver tissue implants used to augment liver functionin cirrhosis patients; (iii) genetically altered cells grown inthree-dimensional culture (such as three-dimensional cultures offibroblasts which express a recombinant gene encoding insulin); (iv) hipprostheses coated with three-dimensional cultures of cartilage; (v)dental prostheses joined to a three-dimensional culture of oral mucosa.

The three-dimensional cultures may be used in vitro to screen a widevariety of compounds, such as cytotoxic compounds, growth/regulatoryfactors, pharmaceutical agents, etc. To this end, the cultures aremaintained in vitro and exposed to the compound to be tested. Theactivity of a cytotoxic compound can be measured by its ability todamage or kill cells in culture. This may readily be assessed by vitalstaining techniques. The effect of growth/regulatory factors may beassessed by analyzing the cellular content of the matrix, e.g., by totalcell counts, and differential cell counts. This may be accomplishedusing standard cytological and/or histological techniques including theuse of immunocytochemical techniques employing antibodies that definetype-specific cellular antigens. The effect of various drugs on normalcells cultured in the three-dimensional system may be assessed. Forexample, drugs that increase red blood cell formation can be tested onthe three-dimensional bone marrow cultures. Drugs that affectcholesterol metabolism, e.g., by lowering cholesterol production, couldbe tested on the three-dimensional liver system. Three-dimensionalcultures of tumor cells may be used as model systems to test, forexample, the efficacy of anti-tumor agents.

The three-dimensional cultures of the invention may be used as modelsystems for the study of physiologic or pathologic conditions. Forexample, in a specific embodiment of the invention, a three-dimensionalculture system may be used as a model for the blood-brain barrier; sucha model system can be used to study the penetration of substancesthrough the blood-brain barrier. In an additional specific embodiment,and not by way of limitation, a three-dimensional culture of mucosalepithelium may be used as a model system to study herpesvirus orpapillomavirus infection; such a model system can be used to test theefficacy of anti-viral medications.

The three-dimensional cell cultures may also be used to aid in thediagnosis and treatment of malignancies and diseases. For example, abiopsy of any tissue (e.g. bone marrow, skin, liver, etc.) may be takenfrom a patient suspected of having a malignancy. If the biopsy cells arecultured in the three-dimensional system of the invention, malignantcells will be clonally expanded during proliferation of the culture.This will increase the chances of detecting a malignancy and, therefore,increase the accuracy of the diagnosis. This may be especially useful indiseases such as AIDS where the infected population of cells is depletedin vivo. Moreover, the patient's culture could be used in vitro toscreen cytotoxic and/or pharmaceutical compounds in order to identifythose that are most efficacious; i.e. those that kill the malignant ordiseased cells, yet spare the normal cells. These agents could then beused to therapeutically treat the patient.

According to the present invention, a relatively small volume of bonemarrow from a diseased patient may be harvested and the patient's bonemarrow destroyed by chemotherapy or radiation. The bone marrow samplemay then be purged of diseased cells using an appropriatechemotherapeutic agent, expanded in vitro, and then readministered tothe patient. In addition to allowing a more effective purge by treatingsmaller volumes of diseased marrow followed by expansion in vitro, thethree-dimensional culture system can be utilized on larger volumes ofpurged marrow. A side effect of most purging agents is destruction anddisruption of normal hematopoietic skin cells, which results in aprolonged time to engraftment and often patient mortality due tosecondary infection. One effective purging agent utilized with acutenonlymphocytic leukemia is 4-hydroperoxyoyolo phosphamide (4HC) whichcauses a two log kill of malignant cells. In traditional treatment, 500ml-1000 ml of diseased marrow is treated by incubation of the marrow exvivo with 60-100 ng of 4HC/ml. Marrow is then cryopreseved and reinfusedinto the patient after 2-3 weeks of clinical chemotherapy. According tothe present invention, a comparable volume of bone marrow may beharvested, purged with 4HC, and then expanded in vitro inthree-dimensional culture, thereby allowing a more rapid engraftmenttime and a decrease in patient mortality.

In vitro methodologies have been useful in reducing rejection of cellsused for transplantation in both animals (bone marrow transplantation inmice) and humans (allogeneic epidermal grafts). The three-dimensionalbone marrow culture can be further used to promote a tolerance of cellsto foreign antigens. In this regard donor hematopoietic cells may begrown in three-dimensional stromal cells from the recipient. Suchcultures may be grown in the presence of three-dimensional thymiccultures which provide additional growth factors and differentiationfactors which will induce maturation of lymphocytes in the bone marrowsystem. As the hematopoietic cells replicate and mature they will beeducated to see the recipient cell antigens as "self", thereby can become tolerant to these "foreign" cells.

Depending upon the intended use for the proliferated cells and tissue,various specialized cells may be added to the three-dimensional culture.For example, the long term growth of bone marrow cells in thethree-dimensional cultures may be enhanced by the addition of certainmononuclear cell populations to the cultures by the addition of growthfactors to the culture medium, or by the use of stromal cellsmanipulated so as to produce a desired growth factor or factors. Cellscollected from these cultures may be used for transfusiontransplantation and banking. The addition of lymphocytes derived from apatient to three-dimensional skin cultures may assist in evaluating anddiagnosing immunological disorders, such as certain autoimmune diseases.Similarly, the addition of lymphocytes and mast cells derived from apatient to three-dimensional skin cultures may assist in evaluating thepatient's allergic response to various allergens without exposing thepatient to the allergens. To this end, the three-dimensional skinculture containing the patient's lymphocytes and mast cells is exposedto various allergens. Binding of lymphocyte-generated IgE to residentmast cells, when "bridged" with the allergen to which the patient issensitive, will result in the release of vasoactive mediators, such ashistamine. The release of such mediators in culture, in response toexposure of the three-dimensional culture to an allergen could bemeasured and used as an indication of the patient's allergic response.This would allow allergy tests to be conducted without exposing theindividual to dangerous and potentially harmful allergens. This systemcould similarly be used for testing cosmetics in vitro.

The three-dimensional culture system of the invention may afford avehicle for introducing genes and gene products in vivo for use in genetherapies. For example, using recombinant DNA techniques, a gene forwhich a patient is deficient could be placed under the control of aviral or tissue-specific promoter. The recombinant DNA constructcontaining the gene could be used to transform or transfect a host cellwhich is cloned and then clonally expanded in the three dimensionalculture system. The three-dimensional culture which expresses the activegene product, could be implanted into an individual who is deficient forthat product.

The use of the three-dimensional culture in gene therapy has a number ofadvantages. Firstly, since the culture comprises eukaryotic cells, thegene product will be properly expressed and processed in culture to forman active product. Secondly, gene therapy techniques are useful only ifthe number of transfected cells can be substantially enhanced to be ofclinical value, relevance, and utility; the three-dimensional culturesof the invention allow for expansion of the number of transfected cellsand amplification (via cell division) of transfected cells.

Preferably, the expression control elements used should allow for theregulated expression of the gene so that the product is synthesized onlywhen needed in vivo. The promoter chosen would depend, in part upon thetype of tissue and cells cultured. Cells and tissues which are capableof secreting proteins (e.g., those characterized by abundant roughendoplasmic reticulum and golgi complex) are preferable. To this end,liver and other glandular tissues could be selected. When using livercells, liver specific viral promoters, such as hepatitis B viruselements, could be used to introduce foreign genes into liver cells andregulate the expression of such genes. These cells could then becultured in the three-dimensional system of the invention.Alternatively, a liver-specific promoter such as the albumin promotercould be used.

Examples of transcriptional control regions that exhibit tissuespecificity which have been described and could be used, include but arenot limited to: elastase I gene control region which is active inpancreatic acinar cells (Swift et al., 1984, Cell 38:639-646; Ornitz etal., 1986, Cold Spring Harbor Symp. Quant. Biol. 50:399-409; MacDonald,1987, Hepatology 7:42S-51S); insulin gene control region which is activein pancreatic beta cells (Hanahan, 1985, Nature 315:115-122);immunoglobulin gene control region which is active in lymphoid cells(Grosschedl et al., 1984, Cell 38:647-658; Adams et al., 1985, Nature318:533-538; Alexander et al., 1987, Mol. Cell. Biol. 7:1436-1444);albumin gene control region which is active in liver (Pinkert et al.,1987, Genes and Devel. 1:268-276); alpha-fetoprotein gene control regionwhich is active in liver (Krumlauf et al., 1985, Mol. Cell. Biol.5:1639-1648; Hammer et al., 1987, Science 235:53-58);alpha-1-antitrypsin gene control region which is active in liver (Kelseyet al., 1987, Genes and Devel. 1:161-171); beta-globin gene controlregion which is active in myeloid cells (Magram et al., 1985, Nature315:338-340; Kollias et al., 1986, Cell 46:89-94); myelin basic proteingene control region which is active in oligodendrocyte cells in thebrain (Readhead et al., 1987, Cell 48:703-712); myosin light chain-2gene control region which is active in skeletal muscle (Shani, 1985,Nature 314:283-286); and gonadotropic releasing hormone gene controlregion which is active in the hypothalamus (Mason et al., 1986, Science234:1372-1378).

In a further embodiment of the invention, three-dimensional cultures maybe used to facilitate gene transduction. For example, and not by way oflimitation, three-dimensional cultures of fibroblast stroma comprising arecombinant virus expression vector may be used to transfer therecombinant virus into cells brought into contact with the stromalmatrix, thereby simulating viral transmission in vivo. Thethree-dimensional culture system is a more efficient way ofaccomplishing gene transduction than are current techniques for DNAtransfection.

In yet another embodiment of the invention, the three-dimensionalculture system could be use in vitro to produce biological products inhigh yield. For example, a cell which naturally produces largequantities of a particular biological product (e.g., a growth factor,regulatory factor, peptide hormone, antibody, etc.), or a host cellgenetically engineered to produce a foreign gene product, could beclonally expanded using the three-dimensional culture system in vitro.If the transformed cell excretes the gene product into the nutrientmedium, the product may be readily isolated from the spent orconditioned medium using standard separation techniques (e.g., HPLC,column chromatography, electrophoretic techniques, to name but a few). A"bioreactor" could be devised which would take advantage of thecontinuous flow method for feeding the three-dimensional cultures invitro. Essentially, as fresh media is passed through thethree-dimensional culture, the gene product will be washed out of theculture along with the cells released from the culture. The gene productcould be isolated (e.g., by HPLC column chromatography, electrophoresis,etc) from the outflow of spent or conditioned media.

Various sample embodiments of the invention are described in thesections below. For purposes of description only, and not by way oflimitation, the three-dimensional culture system of the invention isdescribed based upon the type of tissue and cells used in varioussystems. These descriptions specifically include but are not limited tobone marrow, skin, liver, and pancreas but it is expressly understoodthat the three-dimensional culture system can be used with other typesof cells and tissues. The invention is also illustrated by way ofexamples, which demonstrate characteristic data generated for eachsystem described.

6. THREE-DIMENSIONAL BONE MARROW CULTURE SYSTEM

The three-dimensional culture of the present invention provides for thereplication of bone marrow cells in vitro, in a system comparable tophysiologic conditions. Importantly, the bone marrow cells replicated inthis system include all of the cells present in normal bone marrow,assuming all cell types were present in the original bone marrowinoculum used to initiate the cultures.

Although marrow cells are capable of limited growth when cultured alone,long term growth of these cultures is possible only if stromal cells ortheir secretory products are present. See, Long-Term Bone MarrowCulture, D. G. Wright & J. S. Greenberger, eds., A. R. Liss, New York,(1984) pp. 141-156.

In accordance with the invention, bone marrow cells are grown on athree-dimensional support in co-cultures with stromal cells comprisingfibroblasts (of either fetal or bone marrow origin) or a mixture of celltypes which comprise the stromal components of normal marrow, includingfibroblasts, macrophages, reticular cells, and adipocytes. Factorsderived from media of splenic and/or hepatic (liver) macrophage culturesor from subsets of stromal cells may optionally be added to the culture.The three-dimensional culture system of the present invention appears tomaximize the proliferation of multipotential hematopoietic stem cellswhich have the capability of repopulating bone marrow when the bonemarrow has been destroyed by intrinsically or environmentally-mediateddisease or by the treatment of such disease with chemotherapy and/orradiation.

Using conventional monolayer cell culture techniques, stem cells whichhave marrow repopulating activity (MRA) have been shown to persist andreplicate in long term murine bone marrow cultures. In such systems,however, mature hematopoietic cell expression is limited primarily tothe myeloid and monocytoid lineages. Monolayer cultures of human andnon-human primate bone marrow cells exhibit a steady decline, over time,in assayable progenitors (CFU-GM, CFU-GEMM, BFU-E, etc.). The majormature cell expressed by these monolayer cultures, as in the murinesystem, is the granulocyte. By contrast, hematopoietic progenitors andhematopoietic precursors of all blood cell lineages appear to replicateand proliferate in the three-dimensional stromal system of the presentinvention. Furthermore, differentiation appears to proceed in aphysiologic manner. For example, erythroid, myeloid, lymphoid,macrophagic, and megakaryocytic colonies can continuously arise in thesame culture using the systems as taught by the present invention anddescribed below. Stem cell replication in this system can be inferredfrom the sustained proliferation of committed progenitors.

6.1. OBTAINING BONE MARROW CELLS

Bone marrow cells used in the inoculum may be obtained directly from thedonor or retrieved from cryopreservative storage. The cells are firstseparated from their reticulum by physical means. Accordingly, a smallamount (10-15 cc bone marrow/peripheral blood suspension) may beaspirated from the iliac crest of a donor. For purposes oftransplantation the results of the process are optimal if: (a) theindividual is under 40 years of age at the time his/her marrow is takenfor culture and/or cryopreservation; and (b) the patient isdisease-free; however, the invention is not limited to these criteria.Methods of aspirating bone marrow from a donor are well known in theart. Examples of apparatus and processes for aspirating bone marrow froma donor can be found in U.S. Pat. Nos. 4,481,946 and 4,486,188.

If the bone marrow is to be cultured in order to treat certain patientswith metastatic disease or hematological malignancies, the marrowobtained from the patients should be "purged" of malignant cells byphysical or chemotherapeutic means prior to culturing. At present,physical and chemotherapeutic purging methods require a large samplesize because these methods kill both malignant and normal cellsnonselectively. However, selective methods are currently being developedfor purging. For example, antibodies specific for malignant cells arebeing tested in an attempt to target toxic agents, and specifically killmalignant cells. Such selective purging methods would be efficient ifthe sample size is small. The three-dimensional culture system of theinvention makes this feasible in that a small sample can be purgedefficiently and the remaining healthy cells expanded. The bone marrowremoved from the donor can be replicated or preserved for replication ata later date. If the bone marrow is to be preserved, the bone marrow canbe incrementally frozen using computerized cryotechnological equipment.For example, fresh marrow/blood suspension may be aliquoted in equalvolumes into sterile Nunc tubes and placed in a beaker of crushed iceuntil the cryopreservation chamber is brought to a similar temperature(4° C.). Immediately prior to specimen insertion into the chamber, asolution is added to each Nunc tube using sterile technique, so that thecryoprotectants, dimethylsulfoxide and glycerol, will be present atfinal concentrations of about 7% and 5%, respectively. The freezingprogram is initiated immediately after introduction of the specimen.Freezing program number 1 on the CryoMed Model Number 1010 controller isused.

Using this technique, the cellular viability after freezing and rapidthawing in an 80° C. water bath exceeds 90% as assayed by the trypanblue exclusion method. In addition, greater than 80% of the originalcolony forming unit culture (CFU-C) may be recovered after freezing.Examples of systems for freezing bone marrow and biological substancesin accordance with a precalculated temperature and time curve aredisclosed in U.S. Pat. Nos. 4,107,937 and 4,117,881. Preferably, thebone marrow cells are stored in the liquid phase of liquid nitrogen at atemperature of -196° C. at which temperature all cellular metabolicactivity has ceased.

6.2. ESTABLISHMENT OF THE TEE-DIMENSIONAL STROMAL MATRIX

Stromal cells derived from bone marrow suspensions should be separatedfrom other marrow components. This may be accomplished using anysuitable method known in the art. For example, marrow suspensions may becentrifuged at low forces, e.g., 3000×g for 20 minutes to obtain a whitebase of cells (i.e., the "buffy coat") containing macrophages,fibroblasts, adipocytes, mononuclear blood cells, reticular cells,endothelial cells. The cells of the buffy coat can be suspended in anysuitable medium such as RPMI 1640 medium which may be supplemented withFBS, HS, hydrocortisone hemisuccinate, and appropriate antibiotics.

The cells are then plated onto the three-dimensional matrix. If highconcentrations of stromal cells are used in the inoculum, the stromalsupport matrix will achieve the appropriate degree of subconfluency inshorter time periods. For example, approximately 10⁶ to 10⁷ stromalcells per ml may be plated onto a three-dimensional matrix such assterile nylon mesh (Tetko Corp. of New York, N.Y., USA) contained in apetri dish or other suitable chamber (e.g., Titer-Tek containers).

The inoculated mesh is then placed into a culture flask containing anappropriate volume of nutrient media. The three-dimensional culturesfloat, partially submerged below the surface of the media. The culturesmay be incubated at about 35° C. to 37° C. in about 5% CO₂ in ambientair at a relative humidity in excess of about 90%. Stromal cells whichare predominantly fibroblasts first grow along and completely encircleall of the nylon fibers before beginning to grow into the mesh openings.Depending upon the concentration of cells used in the inoculum, thisprocess may take approximately 5 to 18 days. The degree of subconfluencyof the stromal cells, should be consistent with that seen in FIG. 1prior to the inoculation of hematopoietic cells.

Suspended stromal cells growing in the three-dimensional matrix can becryopreserved using the same technique as previously described for bonemarrow cells. For cryopreservation of sub-confluent cells on the mesh,the nylon mesh may be rolled and inserted into a Nunc tube containingsuitable medium such as RPMI 1640 supplemented with cryoprotectants suchas dimethylsulfoxide and glycerol in final concentrations of about 5%and 15% respectively. Freezing of the stromal cells on the mesh can beaccomplished at initial cooling rates of -1° C./minute from +1° C. to-40° C. A cooling rate of -2° to -3° C./minute is optimum until the endstage temperature of -84° C. is achieved. Approximately 20-25% of thestromal cells may detach from the nylon mesh during this process.

6.2.1. ENHANCING THE GROWTH OF MARROW STROMAL CELLS

The primary rate limiting factor in the growth of marrow stromal cellsis the relatively low mitotic index of the fibroblasts included amongthe marrow stromal cells. The growth of these cells and their depositionof extracellular matrix components may be enhanced by addinghydrocortisone hemisuccinate and/or self-regulating growth factorsderived from the medium of cultured human fetal fibroblasts which have ahigh rate of cell division.

Attachment and growth of fibroblasts on the mesh can also be enhancedby: pre-coating the mesh with solubilized collagen, types I through IV;or using a mesh which is coated or embedded with collagen secreted byfetal human fibroblasts or by adult fibroblasts (hereinafter referred toas "growth enhancing fibroblasts") which have been subsetted based upontheir ability to synthesize certain collagen types. In this regard, thegrowth enhancing fibroblasts are lifted by mild trypsinization from themesh upon reaching confluency (5 to 7 days for fetal human fibroblastsand 14 to 18 days for adult fibroblasts respectively) and may either beinoculated along with stromal marrow cells as previously described orcryopreserved for future use.

In one embodiment of the invention, growth enhancing fibroblasts thatare synthesizing collagen and other extracellular matrix components aregrown on the mesh until they reach subconfluency. A mixture of bothhematopoietic and stromal bone marrow cells are then inoculated onto thesubconfluent growth enhancing fibroblast meshwork.

The methods for growing, subsetting, and cryopreserving growth enhancingfibroblasts are as follows:

(a) Culture of Growth Enhancing Fibroblasts. Any suitable method may beused to culture growth enhancing fibroblasts. For example, fibroblastsmay be grown in suitable medium such as RPMI 1640 supplemented with2-10% FBS or 2-10% HS to which 1 μg/ml hydrocortisone hemisuccinate andantibiotics such as 2 μg/ml gentamycin, penicillin, streptomycin andfungizone have been added. Cultures may be grown at about 5% CO₂ inambient air at 35° C. to 37° C. with a relative humidity in excess ofabout 90%.

(b) Subsetting Growth Enhancing Fibroblasts. A number of methods may beused to subset growth enhancing fibroblasts. For example, about 5.0×10⁶fibroblasts derived from the buffy coat of a bone marrow suspension,dermal fibroblasts, or fibroblasts derived from cadaver livers may beplated onto microtiter wells (1 mm²) and grown to confluency. Thesecells may be lifted from the culture wells by repeated washings, usuallyfour to five times with Hank's balanced salt solution without Ca⁺⁺ orMg⁺⁺. The matrix remaining on the microtiter plates can be examined byindirect immunofluorescence utilizing monoclonal antibodies to variousmatrix components visualized by direct or indirect labels. For example,the binding of unlabeled murine IgG monoclonal antibodies specific for aparticular matrix component can be visualized using enzyme-labeled orfluorescein isothiocyanate-labeled rabbit anti-mouse immunoglobulin G toascertain the collagen types present. A negative selection may then beaccomplished by a number of techniques. For example, the suspended cellsmay be treated with a monoclonal antibody of an isotype that is capableof activating complement (e.g., IgG, IgM, etc.) and which defines aparticular matrix component (e.g., collagen types I through IV, elastin,tropoelastin, or fibronectin) to isolate sub-populations of cellscapable of synthesizing each product. If the cells are then treated withguinea pig complement, those cells to which monoclonal antibody is boundwill be damaged or destroyed. The viable cells remaining in the samplecan be re-plated onto microtiter wells as previously described, grown toconfluency, and lifted. The efficiency of the isolation technique may beverified by examining the matrix secreted by the surviving cells withappropriate monoclonal antibodies visualized by direct or indirectlabeling techniques.

For optimal growth of hematopoietic cells, the initial matrix shouldcontain collagen types III, IV and I in an approximate ratio of 6:3:1.

(c) Cryopreservation of Growth Enhancing Fibroblasts. Growth enhancingfibroblasts can be cryopreserved using the same techniques as previouslydescribed for stromal cells. Like the stromal cells, some of the growthenhancing fibroblasts will also detach from the mesh during freezing.This matrix, however, still contributes to the attachment of marrowstromal cells and therefore diminishes the time required for theestablishment of a matrix conducive to hematopoietic cell growth.

6.3. INOCULATION WITH HEMATOPOIETIC CELLS

Bone marrow cells are suspended in an appropriate nutrient medium (e.g.,RPMI/1640 supplemented with FBS, HS, hydrocortisone, and appropriateantibiotics could be used) and inoculated onto the three-dimensionalstromal support. These cells may either be fresh or derived from aformerly cryopreserved sample which has been rapidly thawed, forexample, in an 80° C. hot water bath. A suitable concentration of cellsare inoculated onto subconfluent stromal cell meshworks. For example,10⁶ to 10⁷ cells can be inoculated onto the three-dimensional stromalmatrices in 25 mm² plastic culture flasks and grown at about 33° C. to34° C. and 5% CO₂ in ambient air. The relative humidity of thesecultures should be in excess of about 90%. After 3 days, the culturetemperature should be raised to about 35° C. to 37° C.

In general, hematopoietic cells will grow in the natural pockets formedby the subconfluent stromal cells and the progenitor cells will remainin the adherent layer of cells. The adherent layer are those cellsattached directly to the mesh or those connected indirectly byattachment to cells that are themselves attached directly to the mesh.Although hematopoietic colonization occurs rapidly, stromal seedingappears to be the rate limiting step for hematopoiesis, since thehematopoietic cells from the inoculum seed mainly those areas where astromal support matrix is present. Colonization occurs in the naturalinterstices formed by the partially developed stromal layers and is alsoseen on the outermost surface of the matrix. The surface colonies aresomewhat smaller than those in the matrix and appear, at times, to bepart of the non-adherent zone. Actually, they are loosely attached andremain after feeding. These cells, which are also found consistently inmonolayer type LTBMC, have been termed the "pseudo-adherent layer"(Coulombel et al., 1983, Blood 62:291-297).

After 4 to 5 days, mature granulocytes, mononuclear cells, anderythrocytes appear in the non-adherent layer as observed by cytospinpreparation. After 7 to 10 days, numerous hematopoietic colonies can beobserved in the interstices of the mesh and are morphologicallyconsistent with CFU-C, mixed colonies, and lymphoid colonies.Megakaryocytic growth is limited but may be observed in this matrix aswell. An average 3.6 cm² culture will produce 450 to 950 CFU-C per week.

Cultures which consist of stromal cells and hematopoietic cells derivedfrom the same individual (autologous) should be fed twice weekly.Cultures which consist of a patient's bone marrow which has beeninoculated onto a stromal cell meshwork derived from anotherindividual(s) (allogeneic) should be fed three times per week to insureadequate depopulation of mature immunocompetent cells from thenon-adherent layer.

6.4. LONG TERM GROWTH OF THREE-DIMENSIONAL BONE MARROW CULTURES

Optionally, the three-dimensional bone marrow cultures may be inoculatedwith mononuclear cells in order to enhance long term growth. Peripheralblood mononuclear cells can be prepared from a heparinized suspensionusing Ficoll-hypaque or Percoll. Peripheral blood cells and bone marrowhematopoietic cells should preferably be derived from the sameindividual (autologous). These may be obtained via venipuncture andcryopreserved at the time the bone marrow specimen is taken. Additionalperipheral blood cells could be procured from the diseased patient ifneeded during the culturing procedure. However, if metastatic disease issuspected, the sample should first be subjected to purging, as mentionedpreviously. The mononuclear cells can be inoculated onto the threedimensional culture soon after the inoculation of bone marrow cells. Forexample, 5×10⁵ to 10⁶ mononuclear cells (the monocyte subpopulation isthe preferred cell type within the mononuclear cell layer for this step)can be inoculated onto meshworks 4 to 5 days after the initialinoculation with bone marrow hematopoietic cells and every third weekthereafter. This procedure may enhance hematopoiesis by 10 to 13% asobserved on a weekly basis.

In our experience, confluent stromal cell cultures will not, or at best,will only poorly support hematopoiesis. Indefinite growth of humanhematopoietic progenitors is possible if they are provided with thenecessary stromal-derived growth/regulatory factors. Thethree-dimensional culturing system of the present invention allows forthe stromal cells to maintain a subconfluent state and thus, produce thefactors necessary for hematopoiesis over long time periods. However, thetime period can be prolonged by further manipulations of thethree-dimensional culture system.

For example, the initial marrow sample may be divided into a number ofaliquots, each containing approximately 10⁶ hematopoietic cells. Each ofthese is inoculated onto a subconfluent stromal cell meshwork. Thecultures may be monitored by direct observation with an inverted phasemicroscope and by differential counts of the non-adherent cells as seenon the cytospin preparation of spent media after each feeding. Prior toreaching confluency, the cultures are treated with collagenase andplaced under mild ultrasonication for approximately 6-10 minutes.

Hematopoietic cells and stromal cells dissociated from the culture canbe fractionated by, for example, density gradient methods. Thehematopoietic cells can be counted using a hemacytometer andapproximately 50% cryopreserved using methods described previously. Theremaining 50% of the hematopoietic cells can be divided into aliquotsconsisting of approximately 10⁶ cells each, and can be inoculated ontosubconfluent stromal cell cultures which have been staggered and grownin parallel. When these begin to reach confluency, the same proceduremay be repeated. This technique: (a) perpetuates the growth ofhematopoietic cells by providing a microenvironment which produces therequired growth factors and, (b) forms a continuous bank wherehematopoietic progenitors may be deposited until the numbers suitablefor engraftment are achieved.

6.5. MODULATION OF HEMATOPOIESIS IN THREE-DIMENSIONAL LONG-TERM BONEMARROW CULTURE

The various cellular components of human marrow can be subcultured inthe three-dimensional system as separate cultures. Macrophages,reticular cells, adipocytes, and fibroblasts may be grown separately andtheir secretory activity modified by treatment with various agents.Modulation of fibroblast activity has been described previously.

Hematopoiesis in long-term human marrow cultures on thethree-dimensional meshwork may also be modulated by secretions ofextramedullary macrophages (Kupffer cells) when grown in culture in thefollowing manner. Kupffer cells can be separated from their organ stromaby, for example, pronase digestion. Briefly, tissue specimens may beincubated for 1 hour in pronase solution [0.2% pronase (Calbiochem) andGeys' Balanced Salt Solution (BSS)] while being gently agitated. The pHof the solution should be maintained at 7.3 to 7.5 using, for example,1N NaOH. Deoxyribonuclease (0.5 mg; Calbiochem) is added at 30 minuteintervals during the above procedure and the resultant cell suspensionis filtered and centrifuged at 350×g for 10 minutes. The pellet may beresuspended in Geys' BSS and the littoral cells (macrophages andendothelial cells) can be separated from the cellular debris and matureblood cells using a Percoll (Pharmacia) gradient. The resultant cellfraction should be washed three time for three minutes each using, forexample, a modified Dulbecco's medium enriched with 10% fetal bovineserum, and plated onto plastic culture dishes at a volume containingabout 3 to 4×10⁶ cells.

After incubation for 1 day, the non-adherent cells are removed bywashing with the culture medium and the adherent cells can be maintainedat 33° C. in a gas mixture consisting of about 6% CO₂ in room air at arelative humidity in excess of about 80%. The growth and/or secretoryactivity of these cells can be stimulated by: (a) varying the CO₂ : O₂ratio, (b) treating the cultures with latex beads, (c) treating thecultures with silica, (d) adding prostaglandin E₂, E₁ or F₂α to themedium, and, (f) supplementing the medium with interleukin 1 orinterleukin 2. Macrophage secretory products may be modulated by theseprocedures/agents.

The medium conditioned with the secretory products of these macrophagesmay be used to modulate the long-term bone marrow cultureerythropoietic/granulopoietic ratio.

6.6. USES OF THE THREE-DIMENSIONAL BONE MARROW CULTURE SYSTEM 6.6.1.TRANSPLANTATION

The three-dimensional bone marrow cultures of the present invention maybe used for treating diseases or conditions which destroy healthy bonemarrow cells or depress their functional ability. The process iseffective especially in the treatment of hematological malignancies andother neoplasias which metastasize to the bone marrow. This aspect ofthe invention is also effective in treating patients whose bone marrowhas been adversely affected by environmental factors, (e.g., radiation,toxins etc.), chemotherapy and/or radiation therapy necessitated by adisease which does not directly affect the bone marrow. In these cases,for example, bone marrow cells from a healthy patient can be removed,preserved, and then replicated and reinfused should the patient developan illness which either destroys the bone marrow directly or whosetreatment adversely affects the marrow.

The three-dimensional culture system of the present invention hasseveral advantages to a patient in need of a bone marrow transplant. Ifthe patient is receiving his or her own cells, this is called anautologous transplant; such a transplant has little likelihood ofrejection. Autologous transplants eliminate a major cause of bone marrowtransplant rejection, that is, the graft vs. host reaction. If themarrow contains malignant or diseased cells, small samples it can bemore effectively purged when using the three-dimensional culture systemof the invention. As previously explained, selective methods for purgingmalignant or diseased cells would work best in small volumes of bonemarrow cells. The three-dimensional culture system described hereinmakes this feasible. Accordingly, a small sample obtained from thepatient can be more efficiently purged using a selective method thatkills malignant cells yet spares healthy cells. The remaining healthycells can then be expanded considerably using the three-dimensionalculture system of the invention. In addition, the process of the presentinvention allows more aggressive treatment of neoplastic disorders withchemotherapeutic agents and radiation. Presently, the extent of thesetreatments is often limited by bone marrow toxicity.

6.6.2. MONITORING A PATIENT'S CONDITION

In a patient with cancer or other diseases, it is often efficacious tomonitor the patient's condition by aspirating a portion of the patient'sbone marrow and examining the sample. In this manner, a metastasis orrecurrence may be detected before it is clinically obvious. Patientswith other conditions that are detectable by examining bone marrow cellsmay also be monitored in this way.

Using the three-dimensional system of the present invention, thelong-term growth of cells derived from an aspirated bone marrow specimenwhich has not been purged enhances the likelihood of the detection ofclonal metastatic cells and hematopoietic cells with chromosomalabnormalities. Such cells would be clonally expanded in thethree-dimensional culture system of the invention and, thus, would bemore easily detected. These cells may escape detection in a conventionalsmear of freshly aspirated (uncultured) bone marrow.

6.6.3. SCREENING COMPOUNDS

The cytotoxicity to bone marrow of pharmaceuticals, anti-neoplasticagents, carcinogens, food additives, and other substances may be testedby utilizing the in vitro bone marrow replication system of the presentinvention.

First, stable, growing cultures of bone marrow cells (including bothstromal and hematopoietic cells) are established. Then, the cultures areexposed to varying concentrations of the test agent. After incubationwith the test agents, the cultures are examined by phase microscopy todetermine the highest tolerated dose (HTD)--the concentration of testagent at which the earliest morphological abnormalities appear.Cytotoxicity testing can be performed using a variety of supravital dyesto assess cell viability in this three-dimensional system, usingtechniques well-known to those skilled in the art. The HTD determinationprovides a concentration range for further testing.

Once a testing range is established, varying concentrations of the testagent can be examined for their effect on viability, growth, and/ormorphology of the different cell types constituting the bone marrowculture by means well known to those skilled in the art.

Similarly, the beneficial effects of drugs may be assessed using thethree-dimensional culture system in vitro; for example, growth factors,hormones, drugs which enhance red blood cell formation, etc. could betested. In this case, stable growing cultures may be exposed to the testagent. After incubation, the cultures may be examined for viability,growth, morphology, cell typing, etc. as an indication of the efficacyof the test substance. Varying concentrations of the drug may be testedto derive a dose-response curve.

Other three-dimensional cell culture systems as disclosed in the presentinvention may be adopted for use in cytotoxicity testing and screeningdrugs. An example of the use of three-dimensional bone marrow culture incytotoxicity assays is presented in Section 18, infra.

7. THREE-DIMENSIONAL SKIN CULTURE SYSTEM

The three-dimensional culture system of the present invention providesfor the replication of epidermal and dermal elements in vitro, in asystem comparable to physiologic conditions. Importantly, the cellswhich replicate in this system segregate properly to formmorphologically and histologically normal epidermal and dermalcomponents.

The use of a three-dimensional co-cultured system for the growth ofepidermal and dermal cells has many advantages over currently usedmonolayer systems. This model allows normal cell-cell interactions andthe secretion of natural growth factors, and the establishment of aconnective tissue network virtually identical to that found in vivo; inparticular, the stromal cells elaborate type-specific andspecies-specific collagen. The resulting completely removable meshworkcan be transplanted, cryopreserved, or used as a target tissue incytotoxicity and drug mechanism studies. In addition, this model allowsfor the growth of fibroblasts alone to form a dermal equivalent, or offibroblasts along with keratinocytes and melanocytes for afull-thickness skin equivalent. All the cells in this three-dimensionalsystem remain metabolically active and undergo mitosis, a majoradvantage over many other models.

The three-dimensional skin culture of the invention has a variety ofapplications ranging from its use as a substrate for screeningcompounds, transplantation and skin grafting, and the study of skindiseases and treatments. For example, the need for thorough testing ofchemicals of potentially toxic nature is generally recognized and theneed to develop sensitive and reproducible short-term in vitro assaysfor the evaluation of drugs, cosmetics, food additives, and pesticidesis apparent. The three-dimensional skin model described herein permitsthe use of a tissue-equivalent as an assay substrate and offers theadvantages of normal cell interactions in a system that closelyresembles the in vivo state.

The need for a skin replacement for burn patients is also evident.Several centers in the United States and Europe have utilized culturedhuman keratinocyte allografts and autografts to permanently cover thewounds of burns and chronic ulcers (Eisinger et al., 1980, Surgery88:287-293; Green et al., 1979, Proc. Natl. Acad. Sci. USA 76:5665-5668;Cuono et al., 1987, Plast. Reconstr. Surg. 80:626-635). These methodsare often unsuccessful and recent studies have indicated that blisteringand/or skin fragility in the healed grafts may exist because of anabnormality in one or more connective tissue components formed under thetransplanted epidermal layer (Woodley et al., 1988, JAMA 6:2566-2571).The three-dimensional skin culture system of the present inventionprovides a skin equivalent of both epidermis and dermis and shouldovercome problems characteristic of currently used cultured keratinocytegrafts. In addition to cytotoxicity and skin replacement, thethree-dimensional skin cultures have applicability to many fields ofindustry including use as a model for studying skin diseases anddeveloping new drugs and treatment modalities, and as a source ofnaturally secreted pharmacologic agents.

7.1. ESTABLISHMENT OF THE THREE-DIMENSIONAL STROMAL SUPPORT ANDFORMATION OF THE DERMAL EQUIVALENT

The inoculation of fibroblasts onto the three-dimensional matrix andtheir growth to subconfluence leads to the formation of a dermalequivalent. In a preferred embodiment of the invention, the fibroblastsare allowed to continue to proliferate until the entire growth substrateis covered; it should be pointed out that even after the fibroblastshave reached confluency, the fibroblasts continue to divide because thethree-dimensional culture permits the exit of cells, thereby preventingcontact inhibition. Although any fibroblasts may be utilized in theinoculum, it is advantageous to use skin fibroblasts, as these willdeposit the appropriate types of collagen and elaborate other dermalcomponents. Fibroblasts may be allogeneic or autologous. Skinfibroblasts may be readily obtained from cellular suspensions preparedby mechanical and/or enzymatic disaggregation of dermal tissue. When thecellular suspension obtained is plated, the fibroblasts will adhere morequickly than other cells, and thus, can be grown to confluence, liftedby mild enzymatic treatment and inoculated onto the three-dimensionalmatrix as previously described.

Once inoculated onto the three-dimensional matrix, adherence of thefibroblasts is seen quickly (e.g., within hours) and the fibroblastsbegin to stretch across the matrix openings within days. Thesefibroblasts are metabolically active, secrete extracellular matrix andrapidly form a dermal equivalent consisting of active fibroblasts andcollagen. Approximately 60% confluency of the fibroblasts on thethree-dimensional matrix is required to support the growth of epidermalcells later inoculated.

While the use of fibroblasts alone is sufficient to form athree-dimensional stromal matrix that functions as a dermal equivalent,additional types of stromal cells may be used to inoculate thethree-dimensional matrix. These include, but are not limited toendothelial cells, pericytes, macrophages, monocytes, lymphocytes,plasma cells, adipocytes, etc.

7.2. INOCULATION OF THE DERMAL EQUIVALENT WITH EPIDERMAL CELLS

In order to culture full thickness skin, i.e., comprising both anepidermal and dermal layer, epidermal cells should be inoculated ontothe dermal equivalent. To this end, melanocytes and keratinocytes may beinoculated simultaneously, or preferably, in sequence. For example,keratinocytes can be inoculated onto subconfluent melanocytes which werepreviously inoculated onto the stromal matrix.

Melanocytes and keratinocytes may be allogeneic or autologous in theirrelationship to fibroblast stromal cells, can be isolated from skinusing known procedures which involve incubating skin in a digestiveenzyme, such as trypsin, in order to separate dermal and epidermallayers.

For example, and not by way of limitation, keratinocytes and melanocytesmay be isolated as follows. A tissue sample, e.g. foreskin, may betrimmed so that the entire surface may be easily exposed to antibiotics.Tissue may be first washed in a concentrated antibiotic solution fortwenty minutes, followed by two subsequent washes of ten minutes each.The outer portion of the tissue may then be cut into small pieces, andthen placed in a 0.15% trypsin solution (in PBS without calcium ormagnesium), quickly removed, placed in a fresh container of the sametrypsin solution (such that all the tissue is covered by solution), andrefrigerated overnight at about 2° C.-8° C. The next day, the tissuepieces may be removed from the trypsin solution, and the epidermisseparated from the dermis using curved. forceps. The epidermis may beplaced in a conical tube, and about 0.15 percent trypsin in PBS (withoutcalcium or magnesium) may be used to digest the tissue into a singlecell suspension; to facilitate this process, the sample my be repeatedlyaspirated into and out of a Pasteur pipette. When the sample appears tobe a single cell suspension, it may be centrifuged at 1400 g for about 7minutes and then resuspended in either growth media or in growth mediacontaining 0.01 mg/ml PMA, which selects for melanocytes. Accordingly,cultures of keratinocytes or melanocytes may be produced. The epidermalcells can be suspended and used to inoculate the dermal equivalent.Alternatively, the epidermal cell suspension can be plated andmelanocytes and keratinocytes separated based upon their differentialattachment qualities. Isolated melanocytes may first be inoculated ontothe dermal equivalent and allowed to grow for a few days prior toinoculation of keratinocytes. This "tissue" grows rapidly and can bemaintained in nutrient media without exogenous growth factors.

A disadvantage of all skin replacements involves the lack of hairfollicles and sweat and sebaceous glands in the transplanted area. Thisdeficiency results in the inability of the patient to regulatetemperature normally and causes the patient to have severely dry skinand pant uncontrollably. To help alleviate this problem biopsies may beremoved from unaffected areas of skin and implanted into the dermalequivalent. By strategically locating these biopsies follicles andassociated glands may be introduced into the transplant site. Biopsiesmay range in size from preferably about 4 cm to 8 cm and may be removedby a standard Baker's punch. Equivalent sized biopsies may then beremoved from the dermal transplant and replaced with follicle-containingimplants, thereby creating a transplanted site which is histologicallynormal and functionally similar to normal skin.

By way of example, and not by limitation, a three-dimensional skin cellculture system may be produced as follows:

(a) fibroblasts are allowed to attach to a mesh and grow for about 7-9days to achieve subconfluence and deposit collagen types I and III, asdescribed previously in regard to the growth enhancing fibroblast usedin the in vitro bone marrow replication system;

(b) melanocytes are plated onto the stromal mesh and are allowed to growto subconfluence for about 5 days;

(c) keratinocytes are inoculated onto subconfluent melanocytes.

In a preferred embodiment of the invention, a three-dimensional skincell culture system may be produced as follows:

(a) fibroblasts are allowed to attach to a mesh and grow for about 14days to achieve confluence and deposit collagen types I and III;

(b) melanocytes are plated onto the stromal mesh and are allowed to growto subconfluence for about 5 days; and

(c) keratinocytes are inoculated onto subconfluent melanocytes.

In particular embodiments of the invention, for example, and not by wayof limitation, in burn patients, it may be advantageous to provide acovering for the wound shortly after injury; in such a situation athree-dimensional cell culture according to the invention consistinglargely of fibroblasts and corresponding to the dermis (and hithertoreferred to as the neodermis) may be placed over the wound, andmelanocytes and keratinocytes may subsequently be applied. The neodermismay comprise-cells autologous or allogeneic to the patient. Epidermalcells may be allogeneic or, preferably, autologous to the patient. Thepresent invention includes the implantation of a living, growingneodermis to which epidermal cells may be added in vivo or in vitro;alternatively, a patient's own cells may be allowed to populate thetransplanted neodermis.

7.3. MORPHOLOGICAL CHARACTERIZATION OF THREE-DIMENSIONAL SKIN CULTURE

Morphological characterization of the three-dimensional stroma indicatethat the fibroblasts inoculated onto the matrix stretch across theopenings, exhibit matrix deposition, and migrate into the interstices ofthe mesh. FIG. 1 illustrates the ability of the fibroblasts to arrangethemselves into parallel layers between the naturally-secreted collagenbundles. These fibroblasts exhibit a rapid rate of cell division andprotein secretion. Melanocytes will grow normally in thethree-dimensional system in that they exhibit dendrite formation, remainpigmented and retain the ability to transfer pigment (see FIGS. 6through 8).

Full thickness skin can be grown in a variety of ways allowing an airinterface. Exposure of the keratinocytes to air promotes a more rapiddifferentiation of keratinocytes and more extensive secretion of keratinlayers, which may be very important in skin penetration studies.

A major advantage of this cell culturing system over others currentlyemployed in dermatological research and engraftment studies is that thefibroblasts in the three dimensional matrix, either subconfluent orconfluent, as described supra, remain metabolically active and secretenatural growth factors and naturally occurring collagen types I and III.The normal metabolic activity of these cells makes this systemparticularly advantageous for use in cytotoxicity assays as well as inthe study of disorders which affect collagen secretion directly, or inwhich an interplay between dermal and epidermal cells results inpathological alterations consistent with the disease.

7.4. TRANSPLANTATION IN VIVO

For purposes of transplantation or engraftment it is preferable to usethree-dimensional matrices constructed of biodegradable materials, e.g.,catgut suture, gelatin, etc. These permit all the advantages of athree-dimensional system but allow a transplanted "tissue" to remainintact while the mesh is naturally degraded and absorbed by the body andreplaced by normal cells migrating into the area.

To form the three-dimensional stromal matrix, it would be preferable toutilize skin fibroblasts obtained from the patient who is to receive thegraft. Alternatively, fetal fibroblasts or a mixture of fetalfibroblasts and the patient's fibroblasts may be used. However,according to the invention, fibroblasts from autologous, allogeneic, orxenogeneic source may be used; Example Section 19 illustrates a specificembodiment of the invention in which human fibroblasts are culturedaccording to the invention, implanted and successfully grafted into pig.More importantly, however, the later inoculated epidermal cells may beadvantageously derived from the patient in order to minimize the risk ofrejection of the graft.

In an alternate embodiment of this aspect of the invention, thethree-dimensional stromal support matrix which forms the neodermis canitself be engrafted onto the patient's wound. In this instance, thepatient's own epidermal cells in the wound area will invade the stromalmatrix and proliferate on the stromal matrix in vivo to form fullthickness skin, i.e., both epidermal and dermal layers. Alternatively,epidermal cells may be seeded onto the neodermis, or sheets of epidermalcells may be applied. Where large wound areas are to be covered, it maybe preferred to engraft the complete three-dimensional skin culture, orto use combinations of both neodermis and full-thickness skin cultures.For example, neodermis could be engrafted at the edges of the wound, andfull thickness cultures in central areas of the wound, to enhance growthand healing and minimize scar formation.

7.5. IN VITRO USES OF THE THREE-DIMENSIONAL SKIN CULTURE

The three-dimensional skin cultures can be maintained in vitro and usedfor a variety of purposes, including screening compounds for toxicity,the study of the mechanism of drug action, the study of skin disordersand disease, etc.

The three-dimensional skin culture could be used as a substrate to testthe cytotoxicity of compounds and other substances. For example, for usein cytotoxicity assays, human cells could be grown onto meshes whichcould be cut into 6 mm disks, places into 96-well flat bottom tissueculture microtest plates, and fed with appropriate medium. The testsubstance could then be added to each sample. The test substance couldbe advantageously applied by limiting dilution technique, in which case,a range of concentrations of the toxic substance can be tested. Eachtissue type may be represented by three rows of meshes in order toprovide data in triplicate. A properly controlled assay could be run asfollows: mesh alone; mesh inoculated with fibroblasts; mesh inoculatedwith fibroblasts and keratinocytes; mesh with fibroblasts andmelanocytes; and mesh inoculated with fibroblasts, melanocytes andkeratinocytes. Chemical agents can be added to each of these substratesand incubated, e.g. for 24 hours. The cytotoxic effect of suchsubstances can be evaluated in a number of ways. For example, aconvenient method, the well known neutral red assay, could be adaptedfor use in this system. To this end, after removal of the medium, eachwell may be rinsed before adding a 0.4% aqueous stock solution ofneutral red dye. After various time intervals the dye is removed andcells are rapidly washed with 4.0% formaldehyde, 1.0% CaCl₂. After about20 minutes, the amount of dye present in each tissue sample can bemeasured by reading absorbance with a Dynatech microplate readerequipped with a 540 nm filter. The amount of vital dye absorbed isdirectly proportional to the number of viable cells present in eachwell. The readings can be averaged and the results expressed asabsorbance observed over baseline levels in control cultures.

Recent studies have indicated that the skin is an integral and activeelement of the immune system (Cooper et al., 1987, The mechanobullousdiseases. In: Dermatology in General Medicine, 3d. Ed., McGraw Hill,N.Y., pp.610-626). One of the major cells in the skin which isresponsible for various immune activities is the Langerhans cell. Thesecells may be prepared from fresh skin samples and added to thethree-dimensional skin culture to produce an immunologically completetissue system. Growth of these cells in the culture for long periods oftime by conventional tissue culture techniques is difficult. The abilityto grow these cells in a three-dimensional system would be of greatimportance in all aspects of study including engraftment, cytotoxicity,and disease mechanisms. This type of skin culture system would have thegreatest impact on research involving auto-immune disorders which havedirect or indirect cutaneous involvement (lupus erythematosis, bullouspemphigoid, etc.).

As explained previously, the three,dimensional skin culture could alsobe used to test for sensitivity to allergens. For allergy tests, theskin cultures could be inoculated with lymphocytes (or plasma cells) andmast cells derived from a patient. Exposure of the culture to anallergen which "bridges" IgE antibodies (produced by the lymphocytes)bound to resident mast cells would result in in the release ofvasoactive mediators such as histamine by the mast cells. The release ofhistamine in the culture could be measured and correlated with theperson's allergic response to the test allergen.

8. THREE-DIMENSIONAL LIVER TISSUE CULTURE SYSTEM

Hepatocytes may be isolated by conventional methods (Berry and Friend,1969, J. Cell Biol. 43:506-520) which can be adapted for human liverbiopsy or autopsy material. Briefly, a canula is introduced into theportal vein or a portal branch and the liver is perfused withcalcium-free or magnesium-free buffer until the tissue appears pale. Theorgan is then perfused with a proteolytic enzyme such as a collagenasesolution at an adequate flow rate. This should digest the connectivetissue framework. The liver is then washed in buffer and the cells aredispersed. The cell suspension may be filtered through a 70 μm nylonmesh to remove debris. Hepatocytes may be selected from the cellsuspension by two or three differential centrifugations.

For perfusion of individual lobes of excised human liver, HEPES buffermay be used. Perfusion of collagenase in HEPES buffer may beaccomplished at the rate of about 30 ml/minute. A single cell suspensionis obtained by further incubation with collagenase for 15-20 minutes at37° C. (Guguen-Guillouzo and Guillouzo, eds, 1986, "Isolated and CultureHepatocytes" Paris, INSERM, and London, John Libbey Eurotext, pp.1-12;1982, Cell Biol. Int. Rep. 6:625-628).

The isolated hepatocytes may then be used to inoculate the threedimensional stroma. The inoculated stroma can be cultured as describedfor bone-marrow and skin in order to replicate the hepatocytes in vitro,in a system comparable to physiologic conditions. This should result inan increased functional expression by the hepatocytes.

Liver cultures maintained in this fashion may be utilized for a varietyof purposes including cytotocity testing, screening drugs, etc. In oneembodiment, three-dimensional liver cultures could be used to screen forcarcinogens and mutagens in vitro. More particularly, it is well knownthat a number of compounds fail to act as mutagens in test organismssuch as bacteria or fungi, yet cause tumors in experimental animals suchas mice. This is due to metabolic activation; i.e., some chemicals aremetabolically altered by enzymes in the liver (the P450 oxidase systemand hydroxylation systems) or other tissues, creating new compounds thatare both mutagenic and carcinogenic. In order to identify suchcarcinogens, Ames and his co-workers devised a screening assay whichinvolves incubating the chemical compound with liver extracts prior toexposure of the test organism to the metabolic product (Ames et al.,1975, Mut. Res. 31:347-364). While a more sophisticated approach, theAmes assay still lacks sensitivity. By contrast, the three-dimensionalliver cultures can be utilized both as the metabolic converters and the"test organism" to determine the mutagenicity or carcinogenicity of thesubstance being tested.

9. THREE-DIMENSIONAL MODEL SYSTEM FOR THE BLOOD-BRAIN BARRIER

According to the invention, a three-dimensional tissue culture modelsystem for the blood-brain barrier may be produced. Briefly, thisthree-dimensional culture recreates the endothelial cell barrier whichseparates the central nervous system from the bloodstream by firstgrowing endothelial cells derived from small blood vessels of the brainto confluence in a three-dimensional mesh. First astrocytes, and thenneurons, are applied to the confluent stromal matrix formed byendothelial cells such that the endothelial cells form a barrier betweenone surface of the culture, above, and the neurons, below. A substanceapplied to the endothelial cell surface must penetrate through theendothelial cell layer to reach the neurons beneath.

For example, and not by way of limitation, endothelial cells may beisolated from small blood vessels of the brain according to the methodof Larson et al. (1987, Microvasc. Res. 34:184) and their numbersexpanded by culturing in vitro using standard methods. These smallvessel endothelial cells may then be inoculated onto a suitable mesh(e.g. the nylon filtration screen made by Tetko, Inc., #3-210/36) andthen grown to complete confluence; silver staining may be used toascertain the presence of tight junctional complexes specific to smallvessel endothelium and associated with the "barrier" function of theendothelium.

Neurons and astrocytes may then be obtained from embryonic or perinatalrats and then separated one from the other using standard techniques(Hattan et al., 1988, J. Cell Biol. 106:. For example, neurons may beseparated from astrocytes by differential adherence to a substrate,astrocytes adhering to dishes precoated with 100 μmg/ml poly-D-lysine,and neurons adhering to dishes precoated with 500 μg/ml poly-D-lysine.

Astrocytes may then be inoculated onto confluent endothelial cellthree-dimensional stromal matrices, cultured for a period of about 5days and then further inoculated with neuronal cells.

The multi-layer three-dimensional tissue culture system comprises onelayer of small blood vessel endothelial cells and another of astrocytesand neurons, and recreates the structure of the blood-brain barrierfound in vivo, wherein substances in the blood must penetrate theendothelium of small blood vessels to reach the neuronal tissue of thebrain. The system can be used to test the ability of substances to crossthe blood-brain barrier. Because many substances are unable to crossthis barrier, there is a long felt need for an in vitro system torapidly screen the penetration abilities of test agents. For example,many antibiotics are unable to cross the blood-brain barrier. It wouldbe useful to be able to rapidly screen newly developed antibiotics fortheir penetration ability; the relatively few antibiotics which may beused to treat central nervous system infections, many of which arerelated to penicillin and therefore associated with the risk of allergicreaction, creates an urgent need for the development of new CNS-activeagents.

10. THREE-DIMENSIONAL PANCREAS TISSUE CULTURE SYSTEM

Suspensions of pancreatic acinar cells may be prepared by an adaptationof techniques described by others (Ruoff and Hay, 1979, Cell Tissue Res.204:243-252; and Hay, 1979, in, "Methodological Surveys in Biochemistry.Vol. 8, Cell Populations." London, Ellis Hornwood, Ltd., pp. 143-160).Briefly, the tissue is minced and washed in calcium-free, magnesium-freebuffer. The minced tissue fragments are incubated in a solution oftrypsin and collagenase. Dissociated cells may be filtered using a 20 μmnylon mesh, resuspended in a suitable buffer such as Hanks balanced saltsolution, and pelleted by centrifugation. The resulting pellet of cellscan be resuspended in minimal amounts of appropriate media andinoculated onto the three-dimensional stroma prepared as previouslydescribed. Acinar cells can be identified on the basis of zymogendroplet inclusions. The culture of pancreatic acinar cells in thethree-dimensional stromal system should prolong cell survival inculture.

11. EXAMPLE: THREE-DIMENSIONAL BONE MARROW CULTURE SYSTEM

The subsections below demonstrate that the three-dimensional culturesystem can be used for the establishment of long term bone marrowcultures for human, non-human primate (macaque), and rat. Thethree-dimensional cultures were evaluated by scanning electronmicroscopy, and the cellular content was evaluated by a number ofmethods. The progenitor content was evaluated by CFU-C and BFU-E, andthe cellular content by differential counts and cytofluorographicanalysis using labeled monoclonal antibodies specific for differenthematopoietic cell lines.

The results indicate that the three-dimensional culture system supportsthe expression of several hematologic lineages as evidenced by thedifferential counts of the non-adherent and adherent zones of the human,macaque and rat cells. Cytofluorographic analysis of the cells attachedto the three-dimensional stroma, i.e., the adherent zone, revealed thepresence of early and late myeloid precursors, mature granulocytes, Band T lymphocytes, megakaryocytes/platelets, and monocytes/macrophages.Although the number of progenitor cells located in the matrix wasvariable, this may have resulted from the random populations of stromalcells used to form the support matrix.

Since hematopoiesis may be dependent on growth-related activities andfactors produced by the support cells, the three-dimensional cultureswere grown in flasks which also contained a confluent monolayer ofstromal cells. An inhibition of both hematopoiesis and stromal cellgrowth in the three-dimensional culture system was observed in thepresence of confluent stromal cells; i.e., the confluent monolayer ofstromal cells in the flask appears to "shut off" the three-dimensionalculture system. When the three-dimensional culture was transferred to anew flask, recovery of hematopoiesis was observed. This result suggeststhat stromal cell products influence not only hematopoietic cells, butother stromal elements as well.

The methods, results and data are described in more detail in thesubsections below.

11.1. PREPARATION OF BONE MARROW SAMPLES 11.1.1. HUMAN BONE MARROW

Bone marrow was aspirated from multiple sites on the posterior iliaccrest of hematologically normal adult volunteers after informed consentwas obtained. Specimens were collected into heparinized tubes andsuspended in 8 ml of RPMI 1640 medium which was conditioned with 10% FBSand 5-10% HS and supplemented with hydrocortisone, fungizone, andstreptomycin. The cell clumps were disaggregated and divided intoaliquots of 5×10⁶ nucleated cells.

11.1.2. NON-HUMAN PRIMATE BONE MARROW

Intact cynomolgus macaque monkey femurs were purchased from the CharlesRiver Primate Center (Porter Washington, N.Y.). The epiphyseal ends ofthe femurs were separated from the bone shaft under sterile conditions.The red marrow was removed, suspended in medium, and divided intoaliquots of 5×10⁶ nucleated cells.

11.1.3. RAT BONE MARROW

Adult male Long-Evans rats (225-400 gm) were anesthetized with ether,and after removal of their femurs, were exsanguinated from the abdominalaorta using heparinized syringes. The femurs were split and the marrowcontents were scraped into a sterile petri dish containing 3 ml ofFischer's medium (Gibco, N.Y.) conditioned with 10% FBS and 10% HS andsupplemented with hydrocortisone, fungizone, heparin, and antibiotics(Naughton et al., 1987, J. Med. 18:219-250). Aliquots of 5-7×10⁶ cellswere prepared.

11.2. ESTABLISHMENT OF THE THREE-DIMENSIONAL STROMAL MATRIX

Nylon filtration screen (#3-210/36, Tetko Inc., N.Y.) was used as atemplate to support all LTBMC described in the examples below. Thescreen consisted of fibers, which were 90 μm in diameter, assembled intoa square weave pattern with sieve openings of 210 μm. Stromal cells wereinoculated using the protocols described in the subsections below.Adherence and subsequent growth of the stromal elements was monitoredusing inverted phase contrast microscopy and scanning electronmicroscopy (SEM).

11.2.1. PREPARATION OF THE SCREEN AND INOCULATION OF STROMAL CELLS FORHUMAN LTBMC

8 mm×45 mm pieces of screen were soaked in 0.1M acetic acid for 30minutes and treated with 10 mM polylysine suspension for 1 hour toenhance attachment of support cells. These were placed in a sterilepetri dish and inoculated with either 5×10⁶ human bone marrow cells orwith equal numbers of human fetal fibroblasts (#GM 1380, CoriellInstitute, New York). Human fetal fibroblasts were grown to confluencein monolayers using RPMI 1540 medium conditioned with 10% FBS, 5-10% HS,supplemented with hydrocortisone, fungizone, and streptomycin, at 35°C., 5% CO₂, and a relative humidity in excess of 90%. These cells werelifted using collagenase (10 μg/ml for 15 minutes) and transferred ontothe screen. After 1-2 hours of incubation at 5% CO₂ the screens wereplaced in a Corning 25 cm² culture flask and floated with an additional5 ml of medium. Screens inoculated with marrow stromal cells weretransferred in a similar manner.

11.2.2. PREPARATION OF THE SCREEN AND INOCULATION OF STROMAL CELLS FORNON-HUMAN PRIMATE LTBMC

Two matrices were employed for LTBMC of monkey cells: nylon meshinoculated with human fetal fibroblasts (as described above) and nylonmesh that was inoculated with 5×10⁶ femoral marrow cells from acynomolgus macaque. Culture conditions and screen pretreatment protocolswere identical to those used for the human cultures described above.

11.2.3. PREPARATION OF THE SCREEN AND INOCULATION OF STROMAL CELLS FORRAT LTBMC

8 mm×45 mm pieces of nylon screen were soaked in 0.1M acetic acid for 30minutes and coated with solubilized type IV mouse collagen (GIBCO Labs,New York) for 1-2 hours. The screen was inoculated with 5-7×10⁶Long-Evans rat femoral marrow cells and after 1-2 hours of incubation in5% CO₂ at 33° C., the mesh was transferred to a 25 cm² culture flask. 5ml of medium was added to float the screen.

11.3. INOCULATION OF THREE-DIMENSIONAL STROMAL MATRIX WITH HEMATOPOIETICCELLS AND ESTABLISHMENT OF CULTURE

When approximately 70% of the mesh openings were bridged with supportcells (10-14 days for rat stroma, 7-13 days for human or monkey stroma,and 4-7 days for human fetal fibroblasts), the screens were transferredto sterile petri dishes and inoculated with 5×10⁶ human or monkeynucleated bone marrow cells or 2-5×10⁶ rat femoral marrow cells,respectively. After 2 hours of incubation in 5% CO₂ each screen wasgently floated in a 25 cm² Corning flask to which 5 ml of medium wasadded. Cultures were fed every 5 days by replacement of the spent mediawith fresh media. The culture vessels were also checked for theappearance of cell monolayers on the walls of the vessels. If suchmonolayers were present at a confluency greater than 25%, thethree-dimensional cultures were transferred to new flasks.

11.4. EVALUATION OF THREE-DIMENSIONAL BONE MARROW CULTURE

The growth of the bone marrow cells and the cell content of thethree-dimensional cultures were assayed histologically, by differentialcounts, CFU-C and BFU-E analysis, and cytofluorographic analysis asdescribed below.

11.4.1. HISTOLOGICAL EVALUATION

For electron microscopic study, cultures were sacrificed at variousintervals following the first inoculation of stromal cells and thesecond inoculation of hematopoietic cells. Briefly, nylon screens werecut into approximately 4 equal parts and were fixed in 3% gluteraldehydephosphate buffer solution, washed, dehydrated in acetone, and placed ina Denton Critical Point Dryer. In some instances, the stromal layer wasphysically disrupted to permit the visualization of the underlying cellgrowth (Naughton et al., 1987, J. Med. 18:219-250). Specimens werecoated with 60% gold and 40% palladium and studied with an Amray SEM.

The growth pattern of human and macaque cells in the three-dimensionalLTBMC was similar to that for rat bone marrow. Briefly, stromal cells(either marrow-derived or fetal human fibroblasts) grew linearly alongand enveloped each nylon strand before starting to span the meshopenings (FIG. 1). Hematopoietic (and stromal) cells of the secondinoculum seed in the natural interstices formed by the stromal cellprocesses which are present in at least 70% of the openings in the 3.6cm² mesh (FIG. 2). Hematopoietic cells did not appear to bind directlyto the nylon but, rather, to those areas where support cells wereattached. Colonization was evident in all cultures by 3-6 days after thesecond inoculation of hematopoietic cells. The 210 μm sieve providedsufficient area for the expression of erythroid, myeloid and othercolonies (FIG. 2) Hematopoiesis was observed on the outer surfaces ofthe nylon screen LTBMC but was most extensive in the interstices of thedeveloping support cells.

11.4.2. TOTAL CELL COUNTS AND CYTOSPIN ANALYSIS OF SPENT MEDIUM OFTHREE-DIMENSIONAL LTBMC

Total cell counts and cytospin preparations were made using spent mediumremoved when the cultures were fed (every 5 days). Cell counts wereperformed using the hemacytometer method. Cytospins were stained withWright's-Giemsa and differential counts were performed on random fields.Analysis of cytospin slides prepared after each feeding revealed thepresence of late stage precursors of the erythroid, myeloid, andlymphoid lineages in the human and monkey cultures (Table II). Thesepersisted for the term of culture of each species tested (39 weeks forthe rat, 12.5 weeks for the primates) although the relative percentagesof the cell types varied. Macrophages/monocytes/fibroblasts releasedinto the non-adherent zone of the human cultures increased with time,mainly at the expense of the myeloid cells (Table II).

                  TABLE II                                                        ______________________________________                                        CELLULAR CONTENT                                                              OF THE NON-ADHERENT ZONE*                                                     Time in                                                                       culture  Differential Count (%)                                               (wk)     MY      E       L     MAC/STR  Other                                 ______________________________________                                        HUMAN                                                                         0        63.9    19.0    10.8  3.6      2.7                                   1        59.0    14.0    8.6   14.9     3.5                                   2        48.5    14.4    9.9   23.7     3.9                                   3        51.9    9.2     9.6   24.7     4.6                                   4        41.2    10.4    6.1   33.9     8.4                                   5        41.9    12.7    10.3  29.0     6.1                                   6        45.2    11.2    8.0   27.2     8.4                                   7        39.8    10.1    6.3   34.8     9.0                                   8        38.6    9.8     6.5   37.1     8.0                                   9        40.3    5.6     6.6   38.4     9.1                                   10       35.9    5.5     6.8   40.6     11.2                                  11       31.3    6.7     5.4   43.2     13.4                                  12       30.1    5.0     4.1   44.6     16.2                                  MACAQUES                                                                      0        64.8    14.2    10.1  8.2      2.7                                   1        60.2    16.0    6.8   12.9     4.1                                   2        57.4    14.9    7.5   16.4     3.8                                   3        49.7    12.4    10.0  23.5     4.4                                   4        49.7    9.9     7.9   26.2     6.3                                   5        43.0    10.7    6.1   32.0     8.2                                   6        39.2    8.0     6.0   36.7     10.1                                  7        ND      ND      ND    ND       ND                                    8        38.8    4.3     8.4   39.2     9.8                                   9        27.6    7.7     8.6   46.1     10.0                                  10       35.5    6.2     7.7   42.0     10.6                                  11       ND      ND      ND    ND       ND                                    12       35.4    6.0     6.9   39.2     12.5                                  ______________________________________                                         *Results reflect an average of 3-5 cultures. Each culture contained one       3.6 cm.sup.2 nylon screen.                                                    MY = myeloid, E = erythroid, L = lymphoid, MAC/STR = macrophages,             monocytes, and fibroblastic cells, Other = megakaryocytes, unidentified       blasts.                                                                       ND = not done.                                                           

11.4.3. TOTAL CELL COUNTS AND CYTOSPIN ANALYSIS OF ADHERENT ZONE OFTHREE-DIMENSIONAL LTBMC

Cell counts of the adherent zone were done at different intervals ofLTBMC by treating the screen with a 1:1 mixture of collagenase andtrypsin (10 μg/ml) and mild ultrasonication. Such analysis of theadherent zone of human and cynomolgus macaque LTBMC revealed that therelative percentage of stromal cells to hematopoietic cells increasedwith time in culture (Table III). In particular, as hematopoieticcolonization proceeded, the relative percentage of stromal elementsdropped. However, stromal cell growth at later periods of the LTBMCoccurs at the expense of hematopoiesis.

                  TABLE III                                                       ______________________________________                                        CELLULAR CONTENT OF THE ADHERENT ZONE*                                        Time in                                                                       culture    Differential Count (%)                                             (wk)       Stromal  E          MY   Other                                     ______________________________________                                        HUMAN                                                                         1          66.4     6.2        20.4 7.0                                       2          60.0     5.4        26.4 8.2                                       3          54.2     6.6        29.2 10.0                                      4          62.6     6.8        24.5 6.1                                       5          65.1     2.7        25.2 7.0                                       6          65.4     6.1        21.6 6.9                                       7          59.7     7.7        25.4 7.2                                       8          64.3     5.1        24.0 6.6                                       9          72.9     2.7        18.4 6.0                                       10         73.2     3.7        17.7 5.4                                       11         71.3     3.0        19.6 6.1                                       12         74.7     2.9        17.4 5.0                                       MACAQUES                                                                      1          53.1     8.0        35.7 3.2                                       2          66.0     8.3        19.2 6.5                                       3          68.6     7.4        18.1 5.9                                       4          57.0     5.1        29.2 8.7                                       5          56.6     5.8        27.5 10.1                                      6          63.1     3.9        24.0 9.0                                       7          ND       ND         ND   ND                                        8          68.1     4.8        20.2 6.9                                       9          59.3     4.0        27.3 9.4                                       10         70.0     4.4        17.3 8.3                                       11         ND       ND         ND   ND                                        12         65.3     4.2        21.9 8.6                                       ______________________________________                                         Cells of the adherent zone were disaggregated by enzyme treatment.            Stroma includes fibroblasts, macrophages, adipocytelike cells, endothelia     E = erythroid; MY = myeloid; Other = lymphoid, thromboid, unidentified        blasts.                                                                       ND = not done.                                                           

Cellular proliferation achieved a steady state condition after severalweeks in culture; similar numbers of cells were found in the adherentand nonadherent zones when the LTBMC were examined on a weekly basis(FIG. 3). The numbers of cells in the non-adherent zone for the first1-2 weeks of culture were somewhat misleading. In our experience, manyof the cells which appear in the medium in the early stages of culturewere formerly loosley attached to the matrix. These become detachedeasily causing an artificially high cell count for the non-adherentzone. Likewise, because of relatively low seeding efficiency, only 5×10⁵to 10⁶ cells initially adhere to the mesh even though the inoculationvolume was 5×10⁶ cells. This "hides" the 2-3 fold cellular proliferationwhich occurs on the mesh during the first week of culture.

11.4.4. CFU-C AND BFU-E CONTENT OF ADHERENT ZONE OF THREE-DIMENSIONALLTBMC

The CFU-C content of the adherent zone of rat LTBMC was determined usinga modification of the method of Bradley and Metclaf (1966, Austr. J.Exptl. Biol. Med. Sci. 44:287-300). Briefly, 4×10⁴ cells were plated andincubated at 37° C. in 7-7.5% CO₂. Pokeweed mitogen rat spleen cellconditioned medium was utilized as a source of colony stimulatingactivity (CSA) for rat CFU-C which were counted after 14 days inculture. Human CFU-C were determined by aliquoting 10⁵ nucleatedcells/ml in Iscove's Modified Dulbecco's medium supplemented with 20%FBS and plating over a layer of 10⁶ PBLs in 0.5% agar (Griffin et al.,1981, J. Clin. Invest. 68:932). Colonies were scored on days 7 and 14after plating (37° C.,7% CO₂). Human BFU-E were assayed after variousintervals of LTBMC in 0.8% methylcellulose in Iscove's medium containing30% FBS, 1% bovine serum albumin, 10⁻ 4 M mercaptoethanol, 2.5-5 I.U./mlof partially purified human urinary erythropoietin (Naughton et al.,1985, J. Surg. Oncol. 30:184-197), and 4.5% ofphytohemagglutinin-stimulated human leukocyte conditioned medium(Cashman et al., 1983, Blood 61:876-884).

Substantial numbers of CFU-C were recovered from the adherent zone ofthe rat and human LTBMC relative to those present in the initialinoculum (FIG. 4). Preliminary findings indicate that BFU-E persisted inthe human LTBMC as well (Table IV).

                  TABLE IV                                                        ______________________________________                                        BFU-E IN THE ADHERENT ZONE AT VARIOUS                                         INTERVALS OF LTBMC                                                            Time of                                                                       culture                                                                       (wk)           Numbers of BFU-E*                                              ______________________________________                                        uncultured marrow                                                                            19 ± 6                                                      2              14 ± 4                                                      4              12 ± 5                                                      7               8 ± 3                                                      9              11 ± 6                                                      10              8 ± 3                                                      ______________________________________                                         *Colonies per 10.sup.5 cells; mean of 3-4 plates ± SEM                

11.4.5. CYTOFLUOROGRAPHIC ANALYSIS OF CELLULAR CONTENT OF ADHERENT ZONEOF THREE-DIMENSIONAL LTBMC

Cytofluorographic analysis of the cellular content of the adherent zonesof human and monkey LTBMC was performed using the EPICS system (CoulterElectronics, Hialeah, Fla.). Cells were separated from the nylon screenat various intervals after the inoculation of hematopoietic cells usingcollagenase and trypsin followed by extensive washing. Then cells wereincubated for 45-60 minutes in Hank's Balanced Salt Solution with Ca⁺⁺or Mg⁺⁺. These were reacted with the following monclonalantibodies-which were conjugated to fluorescein isothiocyanate (FITC):Mo-1, T-3, B-1, Plt-1, and MY-9 (Coulter Immunology, Fla.). MurineIgM-FITC-treated cells were used as controls. Sorting windows werechosen on the basis of fluorescence and light scatter histograms. A0.255 window was appropriately gated and the cellular profiles weredetermined.

Cytofluorographic analysis of adherent zones of the human cultures at 2,7 and 10.5 weeks confirmed the presence of early (MY-9) and late (Mo-1)myeloid cells, B (B-1) and T (T-3) lymphocytes, megakaryocytes/platelets(Plt-1), and monocytes/macrophages (Mo-1) (Table V).

                  TABLE V                                                         ______________________________________                                        MEAN PERCENT REACTIVITY OF                                                    UNCULTURED BONE MARROW AND CELLS                                              FROM THREE-DIMENSIONAL LTBMC WITH                                             MONOCLONAL ANTIBODIES.sup.a                                                   ______________________________________                                        Human.sup.b                                                                        2 wk       7 wk       10.5 wk                                            MAb  LTBMC      LTBMC      LTBMC    Uncultured                                ______________________________________                                        B-1  10.20 ± 1.43                                                                           6.76 ± 0.98                                                                          22.73 ± 1.37                                                                        11.96 ± 1.13                           T-3  18.64 ± 1.88                                                                          11.18 ± 1.86                                                                          13.01 ± 1.84                                                                         9.90 ± 0.64                           Plt-1                                                                               4.40 ± 1.33                                                                           8.08 ± 0.92                                                                          17.05 ± 4.10                                                                         8.72 ± 1.83                           Mo-1 10.10 ± 1.04                                                                          17.26 ± 2.29                                                                          20.98 ± 1.14                                                                         3.46 ± 0.25                           My-9  3.98 ± 0.26                                                                           3.70 ± 0.68                                                                           3.46 ± 0.25                                                                         1.46 ± 0.54                           ______________________________________                                               Macaque.sup.c                                                          MAb      7 wk LTBMC  Uncultured                                               ______________________________________                                        B-1      31.01       8.37 ± 0.99                                           T-3      18.13       11.56 ± 2.1                                           Plt-1    46.50       8.53 ± 1.09                                           Mo-1     40.87       26.64 ± 2.25                                          My-9     21.64       5.49 ± 0.83                                           ______________________________________                                         .sup.a Mean percent reactivity was calculated by subtracting nonspecific      labeling with murineIgM-FITC control. MAb = monoclonal antibody.              .sup.b Results reflect data from 4-5 cultures (±1 SE). Times listed ar     following the inoculation of tissuespecific cells.                            .sup.c Mean of 2 cultures inoculated onto fetal human fibroblasts.       

Human and monkey LTBMC can be established on a stratum of fetal humanfibroblasts but this matrix will not support the growth of rat LTBMC.The fetal fibroblast cells reach a stage of subconfluence which willallow the subsequent inoculation of marrow cells much sooner than marrowstroma. When macaque bone marrow is grown on a bed of fetal fibroblasts,the phenotypic profile of the adherent zone shows that more cells reactwith the Plt-1 antibody than in the other cultures we studied but theother hematologic lineages are represented also (Table V). It is notknown to what extent this finding reflects cross-reactivity of theantibody or a shift in the cell population of the adherent zone mediatedby the fetal cells.

11.4.6. THE EFFECT OF CONFLUENT STROMAL CELL MONOLAYERS ON CELL GROWTHIN THREE-DIMENSIONAL CULTURES

Femoral marrow cells from Long-Evans rats or cynomolgus macaque werepoured through a packed Fenwal wool column as described by Boswell andco-workers (Boswell et al., 1987, Exptl. Hematol. 15:46-53). Briefly,10⁷ -10⁸ femoral marrow cells were placed in 4 ml of medium and pouredover a nylon wool column which was pre-incubated at 37° C. for 45minutes in medium. After an additional 45 minutes of incubation, thenon-adherent cells were drained and the adherent cells were removed byextensive washing and elution with EDTA-Versene solution (1:5000 insaline; GIBCO, Grand Island, N.Y.). Approximately 10⁷ cells wereinoculated in parallel into 25 cm² flasks and grown to 50% and 100%confluence. Pre-established nylon screen LTBMC which were standardizedwith respect to time following the second inoculation, were insertedinto each flask. Growth on the nylon screen LTBMC and the monolayer wasobserved microscopically. Cell counts and cytospin of the nonadherentzone were performed every 5 days. Differential counts of cytospinpreparations of the enzyme dissociated adherent cells were performed 5days after insertion of the nylon screen LTBMC.

When confluent stromal cell monolayers are co-cultured with nylon screenLTBMC, both hematopoiesis and stromal cell growth on the suspendedculture are inhibited (Table VI) as compared to LTBMC suspended inflasks without adherent stroma (p less than 0.05) or with stromal cellsat approximately 50% confluence (p less than 0.05). In addition,co-culture with confluent-stromal monolayers causes the detachment andrelease of mesh-associated stromal cells into the non-adherent zone.Hematopoietic colonies coalesce and cease growing. If the LTBMC istransferred to a new flask, recovery of hematopoiesis is seen by 3-5days.

                  TABLE VI                                                        ______________________________________                                        EFFECT OF STROMAL MONOLAYERS                                                  AT APPROXIMATELY 50% AND 100% CONFLUENCE                                      ON CELLULAR PROLIFERATION IN A SUSPENDED                                      NYLON SCREEN LTBMC IN THE RAT                                                         Time of  MONOLAYER EFFECT                                                     Exposure.sup.b                                                                         ON CELL PROLIFERATION.sup.a                                  Cells     (days)     50% Confluent                                                                             100% Confluent                               ______________________________________                                        Stromal    7           0 ± 2.5                                                                              -13.5 ± 4.7                               Cells.sup.c                                                                             15         +3.3 ± 2.0                                                                             -18.0 ± 5.1                                         28         +1.7 ± 0.9                                                                             -24.3 ± 4.0                               Hematopoietic.sup.d                                                                      7         -1.0 ± 4.1                                                                             -17.3 ± 3.2                               Cells     15         +6.3 ± 3.4                                                                             -30.8 ± 7.7                                         28         +2.7 ± 1.9                                                                              -49.9 ± 10.2                             ______________________________________                                         .sup.a Results are expressed as mean percent differences (+/-) ±1 SEM      as compared to LTBMC grown in the absence of adherent cells on the botton     of the flask. Nylon screen bone marrow cultures were tested at 2 weeks        following the second inoculation (with hematopoietic cells).                  .sup.b Time after introduction of the nylon screen LTBMC into a flask         containing adherent cells at either approximately 50% or 100% confluence.     .sup.c Includes fibroblast, macrophages, adipocytelike cells, endothelia.     .sup.d Includes blasts and late stage precursors of all lineages.        

12. EXAMPLE: THREE-DIMENSIONAL SKIN CULTURE SYSTEM

The subsections below describe the three-dimensional culture system ofthe invention for culturing skin in vitro. Briefly, cultures offibroblasts were established on nylon mesh which had been previouslysterilized. Within 6-9 days of incubation, adherent fibroblasts began togrow into the meshwork openings and deposited parallel bundles ofcollagen. Indirect immunofluorescence using monoclonal antibodies showedpredominantly type I collagen with some type III as well. After 7 days,co-cultures of human melanocytes and kertinocytes were plated onto thefibroblast meshwork. No TPA or cholera toxin was added since trophicfactors are produced by the subconfluent fibroblasts of the adherentlayer. Electron microscopic studies revealed skin cells with normalmorphological characteristics and cell-cell attachments.

12.1. ESTABLISHMENT OF THE THREE-DIMENSIONAL STROMA

Skin fibroblasts were isolated by mincing of dermal tissue,trypsinization for 2 hours, and separation of cells into a suspension byphysical means. Fibroblasts were grown to confluency in 25 cm² Falcontissue culture dishes and fed with RPMI 1640 (Sigma, Mo.) supplementedwith 10% fetal bovine serum (FBS), fungizone, gentamycin, andpenicillin/streptomycin. Fibroblasts were lifted by mild trypsinizationand cells were plated onto nylon filtration mesh, the fibers of whichare approximately 90 μm in diameter and are assembled into a squareweave with a mesh opening of 210 μm (Tetko, Inc., New York). The meshwas pretreated with a mild acid wash and incubated in polylysine andFBS. Adherence of the fibroblasts was seen within 3 hours, andfibroblasts began to stretch across the mesh openings within 5-7 days ofinitial inoculation. These fibroblasts were metabolically active,secreted an extracellular matrix, and rapidly formed a dermal equivalentconsisting of active fibroblasts and collagen. FIG. 1 is a scanningelectron micrograph depicting fibroblast attachment and extension ofcellular processes across the mesh opening.

12.2. INOCULATION OF MELANOCYTES AND KERATINOCYTES

Melanocytes were isolated according to the method of Eisinger and Marko(1982, Proc. Natl. Acad. Sci. USA 79:2018-2022). Briefly, skin sampleswere incubated in trypsin for 4-6 hours, allowing separation of theepidermal and dermal layers. Epidermal cells were suspended in media andplated into 25 cm² Falcon tissue culture flasks. Melanocytes wereseparated from keratinocytes by preferential attachment qualities.Isolated melanocytes were plated onto the fibroblast-coated nylon meshand allowed to grow for 3 days prior to the addition of keratinocytes.Melanocytes grow normally in this system in that they exhibit dendriteformation, remain pigmented, and retain the ability to transfer pigmentto keratinocytes, FIG. 6 depicts the appearance of melanocytes after 3days in the three-dimensional culture system. Isolated keratinocyteswere plated onto the melanocytes after 3-4 days. This "tissue" growsrapidly and is maintained in RPMI 1640, 10% FBS, and the appropriateantibiotics. Since natural growth factors are secreted by the dermalelements, no addition of exogenous factors (e.g., TPA, cholera toxins,etc., as described by Sengel, 1983, in Biochemistry and Physiology ofSkin, Vol. 1, pp. 102-131, Oxford Univ. Press, New York; and Eisinger etal., 1988, Proc. Natl. Acad. Sci. USA 85:1937-1941), is necessary.

12.3. HISTOLOGICAL ANALYSIS OF SKIN CULTURE

The skin cultures were evaluated histologically by light microscopyusing the following procedure: all tissue was fixed in 2.5% bufferedgluteraldehyde, dehydrated in ethanol, and cleared in xylene prior toparaffin embedding. Sections were cut at a thickness of 6 to 8 μm,stained with hematoxylin-eosin and examined for normal and alteredmorphological characteristics.

A cross section of this skin model is shown in the photomicrographs ofFIGS. 7 and 8. Normal cell orientation and morphology is obvious.Epidermal and dermal components completely surround the mesh fiber and adistinct dermal-epidermal junction is evident (FIG. 7). Keratinocytesmanifest a normal morphology and contain pigment granules, and amaturation of cells is seen, with evidence of the formation of a stratumcorneum (FIG. 8).

12.4. TRANSPLANTATION OF THREE-DIMENSIONAL SKIN CULTURE IN VIVO

Our transplantation studies in rats have indicated that thisthree-dimensional system permits the rapid engraftment of the dermal andepidermal components without rejection.

Twenty four rats were employed in the skin transplantation studies.Meshes were cut into 6 mm circular pieces, autoclaved, treated with mildacid, incubated with collagen type IV, incubated with fetal bovine serumand inoculated with stromal cells with or without a second inoculationof keratinocytes. Meshes covered with dermal and/or epidermal cellcomponents were sutured into wound areas and closely examined every12-24 hours as follows: rats received light ether anesthesia and theirdorsal surfaces were shaved and washed with a betadine solution. Four 6mm punches were made with a disposable Baker's punch biopsy needle, andsub-cuticular suturing was used to hold the implanted meshes in place.The rats were closely examined until 12 hours post surgery and thenmonitored every 24 hours.

The areas of mesh implantation showed no signs of erythema, swelling,exudate, or fragility. Meshes were removed at 7 days, 14 days, and 21days post transplantation. Results of these transplants are illustratedin FIGS. 9 and 10. All skin cells are shown 7 days post transplant (FIG.9). FIG. 10 illustrates keratinocytes (k), fibroblasts (f), collagen(c), adipocytes (a) and smooth muscle cells (s) all arranged in anatural configuration around the nylon mesh fiber (m). The absence oflymphocytes and other immune components along with the strong naturalattachment of the cells to the mesh indicates that no rejection istaking place in vivo.

Parallel studies have been performed in which meshes with dermal andepidermal components were implanted into 10 mm×10 mm skin biopsies whichwere then maintained in culture for 14 days and examined histologically.Similar cell migration, attachment, and differentiation patterns wereobserved in these in vitro transplants. The engraftment studies to datehelp to substantiate the hypothesis that our three-dimensional matrixsystem is a true physiologic system in which all cell components areactivated and natural growth factors are being produced.

Although the invention is described in detail with reference to specificembodiments thereof, it will be understood that variations can be madewithout departing from the scope of the invention as described above andas claimed below.

13. EXAMPLE: THREE-DIMENSIONAL LIVER CULTURE SYSTEM 13.1. MATERIALS ANDMETHODS 13.1.1. ANESTHESIA

An adult Long-Evans rat weighing approximately 300 gm. was injectedintraperitoneally with 0.3 ml of injectable sodium pentbarbital.

13.1.2. DISSECTION

The animal was pinched with a sharp forceps to ensure adequateanesthetization. A midline incision was made between the xiphoid processand the inguinal area, followed by further incisions to produce flapspermitting entrance to the abdominal cavity. The intestines and otherorgans were pushed to the right side of the animal, exposing the hepaticportal vein. The hepatic portal vein was further exposed by dissectionand a 4.0 suture was tied around the hepatic portal vein distal to wherethe catheter was to be placed. A second suture was placed around thehepatic portal vein proximal to where the catheter was to be placed. Asmall nick was made in the hepatic portal vein with a 21 gauge needle.The peristaltic pump was turned on and infusion of 500 ml of HEPESbuffer (containing 4.1 gm NaCl, 0.25 gm KCl, 3 ml of 1M NaOH, and 10 mlof 0.24% w/v HEPES stock) was begun; immediately thereafter the inferiorvena cava was cut to allow for the buffer to escape. After infusion wascomplete, the pump was shut off, and the liver was gently removed into aBuckner funnel and then perfused with collagenase solution (0.4 gm NaCl,0.05 g KCP, 10 ml HEPES stock (supra), 0.07 gm CaCl₂.2H₂ O, 6.6 ml 1MNaOH, 50 mg collagenase in 100 cc, brought to a pH of 7.6 at 37° C.).Perfusion was allowed to continue for 15-20 minutes. The contents of theBuckner funnel were filtered, and the liver was removed and placed incollagenase solution containing 1.5% (w/v) BSA.

13.1.3. CELL SOLUTION PREPARATION

The lobes of the perfused liver were separated, and the outer parenchymatrimmed away. The inner parenchyma was then minced in Hanks balancedsalt solution (HBSS) containing physiologic Ca++ and Mg++. Large livertissue fragments were allowed to settle out, and the cell suspension wasthen centrifuged through a Percoll gradient (5 ml of DMEM plus 0.5 Iu/mlinsulin, 0.007 mg/ml glucagon and 20 percent fetal bovine serum) wasplaced in a 50 ml conical centrifuge tube, HBSS plus physiological Ca++and Mg++ was added to the 50 ml. mark, and 5 ml. of Percoll workingsolution [70% stock Percoll plus 30% PBS; stock Percoll was 9 partsPercoll and 1 part 10X concentrated Dulbecco's medium] was layered ontop of the HBSS by centrifugation at 800 g for 10 minutes. Liverparenchyma cells were collected from the bottom of the gradient andadded to three-dimensional mesh cultures with subconfluent stroma.

13.1.4. PREPARATION OF THREE-DIMENSIONAL STROMAL MATRIX

8 mm×45 mm pieces of nylon filtrating screen (#3-210/36, Tetko, Inc.,New York) were soaked in 0.1M acetic acid for 30 minutes and treatedwith 10 mM polylysine suspension for 1 hour. The meshes were placed in asterile petri dish and inoculated with 1×10⁶ fibroblasts collected fromrat liver in DMEM complete medium. After 1-2 hours of incubation at 5%CO₂ the screens were placed in a Corning 25 cm² tissue culture flask,floated with an additional 5 ml. of medium, and allowed to reachsubconfluence, being fed at 3 day intervals.

13.1.5 MAINTENANCE OF THREE-DIMENSIONAL LIVER TISSUE CULTURES

After inoculation of liver parenchymal cells onto the three-dimensionalstromal matrix, cultures were maintained in DMEM complete medium at 37°C. and 5% CO₂ in a humidified atmosphere and were fed with fresh mediumevery 3 days.

13.2. RESULTS AND DISCUSSION

Adult liver cells cultured in this fashion exhibited active mitosis andcontinued to secrete proteins over a three-week period of time.Hepatocytes oriented themselves into cords of cells (FIG. 11 andhistologically resembled hepatoblasts or regenerating liver cells duringthe first 10-12 days of three-dimensional culture (FIG. 12). As thecultures became highly confluent, the parenchymal cells began toresemble mature adult hepatocytes with bile duct cells, Kupfter cellsand other liver stromal cells still present. Cells divided approximatelyevery 24 hours for the first 10-12 days of culture and continued todivide every 72 hours for up to a three-week period. Albumin secretioncontinued over the three-week culture period and hepatocyte retainedtheir activated enzymes which allowed them to metabolize products invitro. After cultures reached full confluency they could be maintainedas viable substrates for up to 12 weeks.

14. EXAMPLE: THREE-DIMENSIONAL MUCOSAL EPITHELIUM TISSUE CULTURE SYSTEM14.1. MATERIALS AND METHODS 14.1.1. PREPARATION OF MUCOSAL EPITHELIALCELLS

Samples of oral mucosal tissue were obtained from orthodontic surgicalspecimens. Tissue was washed three times with fresh MEM containingantibiotics (2 ml of antibiotic antimycotic solution, from GIBCO, Cat.#600-5240 AG; and 0.01 ml of gentamycin solution from GIBCO, Cat.#600-5710 AD per 100 cc MEM), cut into small pieces, then washed with0.02% EDTA (w/v). 0.25% trypsin (in PBS without Ca++ or Mg++ was added;after a few seconds, the tissue pieces were removed and placed in fresh0.25% trypsin (in PBS without Ca++ or Mg++) and refrigerated at 4° C.overnight. Tissues were then removed and placed in fresh trypsinsolution, and gently aggitated until cells appeared to form asingle-cell suspension. The single-cell suspension was then diluted inMEM containing 10% heat-inactivated fetal bovine serum and centrifugedat 1400 g for 7 minutes. The supernatant was decanted and the pelletcontaining mucosal epithelial cells was placed into seeding medium.Medium consisted of DMEM with 2% Ultrosen G, 1×L-glutamine,1×nonessential amino acids, penicillin and streptomycin. The cells werethen seeded onto a three-dimensional stromal matrix (see infra).

14.1.2. PREPARATION OF THE THREE-DIMENSIONAL STROMAL MATRIX

The three-dimensional stromal matrix used in mucosal epithelium cultureswas generated using oral fibroblasts and 8 mm×45 mm pieces of nylonfiltration screen (#3-210/36, Tetko Inc., New York) as described abovefor three-dimensional liver cultures in Section 13.1.4).

14.1.3. MAINTENANCE OF THREE-DIMENSIONAL MUCOSAL EPITHELIUM TISSUECULTURES

After inoculation of mucosal epithelial cells onto the three-dimensionalstromal matrix, cultures were maintained in DMEM complete medium at 37°and 5% CO₂ in a humidified atmosphere and were fed with fresh mediumevery 3 days.

14.2. RESULTS AND DISCUSSION

FIG. 13 is a photomicrograph of a cross-section of a three-dimensionalmucosal epithelium tissue culture produced by the methods describedsupra. The tissue culture was found to recapitulate the stratifiedsquamous epithelium of the oral mucosa in vivo; note that as the cellsapproach the surface of the culture, the nuclei become flattened andoriented in a plane parallel to the surface, as occurs in vivo.

15. EXAMPLE: THREE-DIMENSIONAL PANCREAS TISSUE CULTURE SYSTEM 15.1.MATERIALS AND METHODS 15.1.1. PREPARATION OF PANCREATIC ACINAR CELLS

Pancreatic acinar cells were prepared by an adaptation of the techniquedescribed in Ruoff and May (1979, Cell Tissue Res. 204:243-252) and Hay(1979 in "Methodological Surveys in Biochemistry", Vol. 8, CellPopulations," London, Ellis Hornwood, Ltd. pp. 143-160). The tissue wascollected from adult male Long-Evans rats and minced and washed incalcium free, magnesium free HBSS buffer. The minced tissue was thenincubated in a solution containing 0.25 percent rypsin and collagenase.Dissociated cells were filtered using a 20 μm nylon mesh, resuspended inHBSS, and pelleted by centrifugation at 300 g for 15 minutes. Theresulting pellet was resuspended in a small amount of DMEM completemedium and inoculated onto three-dimensional stroma (see infra).

15.1.2. PREPARATION OF THE TEE-DIMENSIONAL STROMAL MATRIX

The three-dimensional stromal matrix used in pancreatic tissue cultureswas generated using adult rat pancreatic fibroblasts and 8 mm×45 mmpieces of nylon filtration screen (#3-210/36, Tetko, Inc., New York) asdescribed above for three-dimensional liver cultures in Section 13.1.4.

15.1.3. MAINTENANCE OF THREE-DIMENSIONAL PANCREATIC TISSUE CULTURES

After inoculation of pancreatic acinar cells onto the three-dimensionalstromal matrix, cultures were maintained in DMEM complete medium at 37°C. and 5% CO₂ in a humidified atmosphere and were fed with fresh mediumevery 3 days.

15.2. RESULTS AND DISCUSSION

FIG. 14 is a photomicrograph of a cross-section of a three-dimensionalpancreas tissue culture produced by the methods described supra. Thetissue culture acinar cells may be identified on the basis of zymogendroplet inclusions [arrow], as compared to the more homogeneousappearance of stromal cells (asterisk). Islet cells remain concentratedin the center of each mesh opening and form a structure containing1-2×10⁵ insulin-secreting cells.

16. EXAMPLE: TEE-DIMENSIONAL MODEL SYSTEM FOR THE BLOOD-BRAIN BARRIER16.1. MATERIALS AND METHODS 16.1.1. PREPARATION OF SMALL VESSELENDOTHELIAL CELLS

Small vessel endothelial cells isolated from the brain according to themethod of Larson et al. (1987, Microvasc. Res. 3.4:184) were cultured invitro using T-75 tissue culture flasks. The cells were maintained inDulbecco's Modified Eagle Medium/Hams-F-12 medium combination (thesolution is available as a 1:1 mixture). The medium was supplementedwith 20% heat-inactivated fetal calf serum (FCS), glutamine, andantibiotics. The cells were seeded at a concentration of 1×10⁶ cells perflask, and reached a confluent state within one week. The cells werepassaged once a week, and, in addition, were fed once a week withDMEM/Hams-F-12 containing FCS, glutamine, and antibiotics as describedsupra. To passage the cells, flasks were rinsed twice with 5 ml of PBS(without Ca++ or Mg++) and trypsinized with 3 ml of 0.05% Trypsin and0.53 mM EDTA. The cells were pelleted, resuspended, and tested forviability by trypan blue exclusion, seeded and fed with 25 ml of theabovementioned DMEM/Hams-F-12 supplemented medium. A factor VIII relatedantigen assay (Grulnick et al., 1977, Ann. Int. Med. 86:598-616) is usedto positively identify endothelial cells, and silver staining was usedto identify tight junctional complexes, specific to only small vesselendothelium.

16.1.2. PREPARATION AND SEEDING OF MESH

Nylon filtration screen mesh (#3-210/36, Tetko, Inc., New York) wasprepared essentially as described above for liver, pancreas, bonemarrow, etc. tissue culture systems. The mesh was soaked in an aceticacid solution (1 ml glacial acetic acid plus 99 ml distilled H₂ O) forthirty minutes, was rinsed with copius amounts of distilled water andthen autoclaved. Meshes were coated with 6 ml fetal bovine serum per 8×8cm mesh and incubated overnight. The meshes were then stacked, threehigh, and 3×10⁷ small vessel endothelial cells (cultured as describedsupra) were seeded onto the stack, and incubated for three hours at 37°C. under 5% CO₂ in a humidified atmosphere. The inoculated meshes werefed with 10 ml of DMEM/Hams-F-12 medium every 3-4 days until completeconfluence was reached (in approximately two weeks).

16.1.3. PREPARATION OF NEURON AND ASTROCYTE CELL POPULATIONS

Neurons and astrocytes were isolated from fetal rat cerebellum. Thecerebellums from 5 rats were dissected out and placed in PBS buffer. ThePBS was then removed and 1 ml of trypsin solution (10 mg trypsin, 1 mlPBS with 0.01 g MgSO₄ -7H₂ O, and 6 μl 1N NaOH) was added to each. After3 minutes, the tissue was rinsed with about 1ml PBS buffer and 2 ml of astock solution consisting of 7.5 mg DNAse plus 15 ml Earles BME).Individual cells were then brought into suspension by aspirating tissuethrough progressively smaller syringe needles ranging from 18 to 25gauge, until the solution was cloudy. The resulting single-cellsuspension was then centrifuged at 800 g for 5 minutes, and the cellpellet resuspended in medium and then passed through a 33 μm filter.Cells were then layered onto a 60%/35% Percoll step gradient andcentrifuged for 10 min. at 800 g. Cells at the 0%/35% interface weremostly glia and astrocytes; cells at the 35%/60% interface were largelyneurons. Both populations were collected and diluted separately in 5 mlof PBS, washed, and collected by centrifugation at 2500 g for 5 minutes.The cells were then resuspended in medium (BME containing 10%heat-inactivated horse serum). Both cell types were, separately, platedonto culture dishes precoated with poly-D-lysine. First they were platedonto dishes precoated with 100 μg poly-D-lysine, incubated for 20-45minutes, and then lightly rinsed with PBS; glia and astrocytesselectively adhered to the culture dishes, and neurons were rinsed off.The rinse buffer was then plated onto culture dishes coated with 500 μgpoly-D-lysine, in which case neurons adhered to the culture dishes.

16.1.4. SEEDING THE ASTROCYTES ONTO THREE-DIMENSIONAL ENDOTHELIAL CELLCULTURES

5×10⁵ astrocytes were seeded onto meshes covered with confluentendothelial cells (described supra) by removing the medium from themesh, inoculating the meshes with the astrocytes, and then incubatingfor one hour at 37° C. and 5% CO₂ in a humidified atmosphere. The meshwas then fed with DMEM-k12 containing interferon, transferrin, selenium,and subsequently fed at 2-3 day intervals.

16.1.5. SEEDING NEURONS ONTO THREE-DIMENSIONAL ENDOTHELIALCELL-ASTROCYTE TISSUE CULTURES

After approximately 5 days, neurons were seeded onto the endothelialcell-astrocyte tissue cultures. Neuronal cell cultures, exhibitingneurite outgrowth (which was observed after about one week in culture),were harvested and approximately 5×10⁵ cells were seeded onto theendothelial cell/astrocyte three-dimensional culture meshes. Neuronalcells were seeded in a minimal volume of culture medium, and thenincubated for 3 hours at 37° C. and 5% CO₂ in a humidified atmosphere,after which time meshes were fed with a standard volume of DMEM/F12,reincubated, and subsequently fed at 2-3 day intervals.

16.2. RESULTS AND DISCUSSION

Nylon mesh was precoated with fetal bovine serum, onto which smallvessel endothelial cells, grown to confluence in standard monolayerculture, were seeded and grown to complete confluence.

Neurons and astrocytes were prepared from the cerebellum of fetal rats,and separated by differential adherence. Astrocytes were grown on theconfluent endothelial cell three-dimensional stromal matrix, and,subsequently, neuronal cells were added to the three-dimensional tissueculture.

The resulting endothelial cell/astrocyte/neuron three-dimensional tissueculture, was then maintained until it reached a second stage ofsemi-confluence covering the layer of endothelial cells. Thismulti-layer three-dimensional tissue culture system, as shown in FIG.15, wherein one layer consists of confluent small blood vesselendothelial cells and the other layer consists of astrocytes andneurons, recreates the structure of the blood-brain barrier found invivo, wherein substances in the blood must penetrate the endothelium ofsmall blood vessels to reach the neuronal tissue of the brain. Such ablood-brain barrier model system can be used to study the passage, orlack thereof, of chemicals or viruses into the brain; it is advantageousto determine which antibiotics, or antivirals for example, can penetratethe blood-brain barrier to treat central nervous system infections.Further, such a model system can be used as a substrate for the study ofthe action and potency of various neurotoxins.

17. EXAMPLE: TEE-DIMENSIONAL ADENOCARCINOMA TISSUE CULTURE SYSTEM 17.1.MATERIALS AND METHODS 17.1.1. PREPARATION OF ADENOCARCINOMA STROMAL ANDPARENCHYMAL CELLS

Adenocarcinoma cells were separated from stromal cells by mincing tumorcells in HBSS, incubating the cells in 0.27% trypsin for 24 hours at 37°C. and further incubating suspended cells in DMEM complete medium on aplastic petri dish for 12 hours at 37° C. Stromal cells selectivelyadhered to the plastic dishes.

17.1.2. PREPARATION OF THE THREE-DIMENSIONAL STROMAL MATRIX

The three-dimensional stromal matrix used in adenocarcinoma tissuecultures was generated using stromal cells derived from the tumor (seeSection 17.1.1., supra) and 8 mm×45 mm pieces of nylon filtration screen(#3-210/36, Tetko, Inc., New York), as described above forthree-dimensional liver cultures in Section 13.1.4.

17.1.3. MAINTENANCE OF THREE-DIMENSIONAL ADENOCARCINOMA TISSUE CULTURES

After inoculation of adenocarcinoma cells onto the three-dimensionaltumor stromal matrix, cultures were maintained in DMEM complete mediumwith high glucose, 15% FBC and 0.03% glutamine at 37° C. and 5% CO₂ in ahumidified atmosphere and were fed with fresh medium every 3 days.

17.2. RESULTS AND DISCUSSION

FIG. 16 is a photomicrograph of a three-dimensional adenocarcinomatissue culture. Adenocarcinoma cells showed a characteristic piling andorientation into a three-dimensional tumor-like structure. Cellsretained their epithelial-like appearance.

18. EXAMPLE: THREE-DIMENSIONAL TISSUE CULTURE CYTOXICITY TESTING SYSTEM18.1. MATERIALS AND METHODS 18.1.1. PREPARATION OF THREE-DIMENSIONALBONE MARROW TISSUE CULTURES

Three-dimensional bone marrow tissue cultures were prepared according tothe method outlined in Section 11, supra.

18.1.2. EXPOSURE OF THREE-DIMENSIONAL BONE MARROW CULTURES TO CYTOTOXICAGENTS

Individual three-dimensional bone marrow cultures were maintained ineach well of a 96 well tissue-culture tray for cytotoxicity testing.

Cultures were exposed to 10-fold serial dilutions of adriamycin, rangingfrom 0.1 TO 10 μm, for 24 hours. Controls were exposed to ten-foldserial dilutions of bovine serum albumin (BSA).

Similarly, other three-dimensional bone marrow cultures, in a 96 wellmulti-well tissue culture unit, were exposed to ten-fold serialdilutions of cis-platinum, ranging from 1-75 μm for 24 hours. Controlswere exposed to serial dilutions of BSA.

In all cases, monolayers of human fibroblasts, cultured usingconventional techniques, were compared to three-dimensional cultures ofeither stromal cells alone, or in conjunction with hematopoietic cells.

18.1.3. CYTOTOXICITY ASSAY

Media was removed from cells, and 0.2 ml of neutral red dye-mediasolution (see Section 18.1.4, infra) was added to each well. Thecultures were then incubated at 37° C. for three hours. In culture trayscontaining three-dimensional cultures, well 1A served as the control andcontained mesh alone without cells.

After incubation, dye/medium was removed, and each well was washedrapidly with formal-calcium (see 18.1.4, infra) to remove unincorporatedneutral red and enhance attachment of the cells to the substratum.

0.2 ml of acetic acid/ethanol solution (see 18.1.4, infra) was added toeach well and the cultures were kept at room temperature for 15 minutes(to extract the dye) and then shaken for a few seconds on a shakerplate.

Culture trays were then transferred to a Dynatech microplate readerequipped with a 540 nm filter for automated spectrophotometric readingand recording. Acetic acid/ethanol solution in a control well served asa blank.

18.1.4. SOLUTIONS FOR CYTOTOXICITY ASSAY

Neutral red/medium was prepared as follows. Neutral red was prepared asa 0.4% aqueous stock solution; and was shielded from light by foil. Afresh 1:80 dilution of the dye was made. Immediately before use, the dyemedium solution was centrifuged for 5 minutes at 1500 g and thesupernatant fluid was used for the neutral red assay.

Formal-calcium was prepared as follows. 5 g of CaCl₂ (anhydrous) wasadded to 497.5 ml of sterile distilled H₂ O. 2.5 ml of 40% formaldehydewas then added to produce a formal-calcium solution which was 1% CaCl₂and 0.5% formalin.

Acetic acid ethanol solution was produced as follows. 1.09 ml glacialacetic acid was added to 99 ml of 50% ethanol.

Adriamycin and cis-platinum were obtained from Sigma Chemical Co., St.Louis, Mo.

18.2. RESULTS AND DISCUSSION

FIGS. 17 and 18 show the results of three-dimensional bone marrowculture cytotoxicity assays, using adriamycin and cis-platinum,respectively, as test agents. Note that, in each case, thethree-dimensional culture systems show a dose-related response to testagent. Significantly, with either adriamycin or cis-platinum, the TD₅₀for bone marrow three-dimensional cultures was different from the TD₅₀determined using conventional fibroblast monolayer cultures.Importantly, these results indicate that monolayer cultures may not beaccurate measures for cytotoxicity; perhaps because the cells aregrowing in an extremely unnatural environment, monolayer cell culturesmay be more sensitive to toxic agents. It is crucial to be able todetermine the actual toxicity of a test substance; for example, inchemotherapy, it may be important to administer the highest dosetolerable in order to effectively eliminate malignant cells.Underestimating the highest tolerated dose may result in administering aless effective amount of anti-tumor agent. By providingthree-dimensional tissue cultures not only of bone marrow and othernormal tissues, but tumor tissues as well, the present invention enablesthe in vitro determination of the optimal dose of chemotherapeuticagent.

19. EXAMPLE: THREE-DIMENSIONAL SKIN CULTURE SYSTEM FOR IMPLANTATIONUSING A NEODERMIS IN MICROPIGS

Skin transplants were performed on four Charles River micropigs.Experiments were designed to compare the effects of neodermis, meshsubstrate permeated with cell lysate, and mesh alone on the contraction,healing and epithelialization of split-thickness and full-thicknesswounds. Multiple parallel wounds were compared along the dorsal surfaceof each animal to allow accurate assessment of healing in each area. Inthese studies a biodegradable mesh was seeded with pig dermalfibroblasts and transplanted as a dermal replacement. Other meshesincluded human dermal fibroblasts and pig dermal fibroblasts seeded withpig keratinocytes. By monitoring the engrafted areas throughhistological sections and gross changes in appearance (exudate,erythema, etc.), we were able to study the efficacy of thethree-dimensional skin system as a transplant modality.

19.1. MATERIALS AND METHODS 19.1.1. PREPARATION OF THE WOUND BED

For proper evaluation of the epidermal graft it was essential that thewound graft bed be prepared so that no dermis, hair follicles, sweat orsebaceous glands remained. To achieve this, a Browne dermatome at asetting of 0.075-0.090 inch was used to remove full thickness skin fromthe upper lateral side of the pig. Wound areas of 5 cm×5 cm werecreated. When lower lateral regions were to be prepared, the setting wasadjusted to 0.60-0.075 inches. The mesh alone or mesh with culturedcells was placed on the bed, just above the fascia. The steriledermatome,prepared bed reduced the possibility of contamination andallowed for absolute hemostasis in the graft bed.

If minor bleeding occurred after the skin removal, a dry gauze dressingwas placed on the wound and pressure was applied for 10-15 minutes. Atall times the sterile bed was covered with sterile gauze pre-wetted withPBS until the grafts were placed on the fascia. Silk sutures (3-0) wereplaced approximately one and one-half inches apart on both sides of theprepared graft bed to hold a compression stent in place. Cultured grafts(meshes with cells) were removed from tissue culture flasks with sterileforceps and sutured into place using conventional subcuticular stitches.Grafts were covered with petrolatum gauze and silk ligatures were tiedso as to provide a compression stent. The pig was bandaged withElastoplast. Wound dressings were changed in four days.

19.1.2. ANESTHESIA

Pigs were anesthetized with the use of ketamine hydrochloride (Ketalar)and were kept under anesthesia by a mixture of halothane, nitrous oxide,and oxygen. The skin area was washed with povidone-iodine (Betadine) and7% alcohol, prior to preparation of four full-thickness graft beds onthe lateral side of the pig, as described above.

19.1.3. ANIMAL MAINTENANCE

After 4 to 5 days the wound dressing was removed, grafted areas werestudied for signs of infection and/or rejection, and then were coveredonce more with petrolatum gauze and bandaged with Elastoplast. Woundswere subsequently observed at four-day intervals with biopsies beingtaken from treated areas. Animals were anesthetized with Ketalar toallow sterile removal of a 4 mm biopsy utilizing a disposable Baker'spunch and sterile technique. Samples were sent for histologicalevaluation in order to assess graft attachment and wound healing.

19.1.4. EPITHELIAL GRAFTS

Selected animals received half-grafts of autologous keratinocytes 10days after implantation with the dermal equivalent. Keratinocyte sheetsproduced from isolated cells grown to over-confluence had been culturedfor 10-14 days before implantation onto the dermal equivalent. Sheetswere attached by four topical sutures and covered with petrolatum gauzeand bandaging to allow cell attachment.

19.2. RESULTS

In all animals treated, dermal equivalents attached well, preventedcontraction and dehydration, and provided a living tissue bed onto whichepithelial cells could migrate or be placed in an autologous transplant.FIG. 19 is a representative of healing 10 days after implantation ofhuman dermal equivalent (neodermis) into a full thickness wound. Wenoted a minimal contraction of the area and no signs of rejection,indicating the utility of allogenic fibroblasts in transplants.Histological evaluation of these areas shows active growth offibroblasts and deposition of collagen with minimal white cellinfiltration (FIG. 20). Split thickness wounds showed remarkabledifferences in contraction, epithelialization, pigmentation, and hairgrowth when comparing wound treated with dermal equivalent (left) andbiodegradable mesh alone (right) (FIG. 21). Mesh soaked with fibroblastcell lysate showed an enhancement of epithelial growth around mesh fiber(FIG. 22), but an overall slower healing progress than in areas treatedwith living neodermis.

Neodermis enhanced epithelial migration onto the healing area. As seen,in FIG. 23, deep rete pegs are formed by the keratinocytes as theymigrate onto and attach to the living dermal equivalent. This pattern ischaracteristic of epithelialization in healing areas.

Autologous keratinocytes attached well and had even healing onto theneodermis. FIG. 24 illustrates the comparison of healing of awound--half of which has received an autologous epithelial graft andhalf of which received neodermis alone. The epithelial graft healedevenly, prevented further contraction, and firmly attached to theunderlying dermal equivalent. FIG. 25 shows the even growth andattachment of the epidermal cells to the neodermis. The neodermisappeared to consist of actively growing fibroblasts andnaturally-secreted fibroblasts. The mesh fibers were still present asseen in cross-section.

19.3. DISCUSSION

Transplantation experiments to date have indicated that the neodermis(fibroblasts and naturally secreted collagen on the biodegradable mesh)provides an excellent treatment for full-thickness wounds. Successfuluse of xenogeneic transplants illustrates the ability to utilizeallogeneic neodermis in burn victims and patients with decubitus ulcers.The transplants allow migration of epithelial cells onto the implantedsurface as well as support and growth of autologous epithelial sheets.Grafts were permanent, with no evidence of either superficial or deepscarring after four months.

What is claimed is:
 1. A three-dimensional pancreatic cell culture comprising pancreatic acinar cells cultured on a living stromal tissue prepared in vitro, said living stromal tissue comprising stromal cells and connective tissue proteins naturally secreted by the stromal cells attached to and substantially enveloping a framework composed of a biocompatible, non-living material formed into a three-dimensional structure having interstitial spaces bridged by the stromal cells.
 2. The three-dimensional cell culture of claim 1 in which the stromal cells are fibroblasts.
 3. The three-dimensional cell culture of claim 1 in which the stromal cells are a combination of fibroblasts and endothelial cells, pericytes, macrophages, monocytes, leukocytes, plasma cells, mast cells or adipocytes.
 4. The three-dimensional cell culture of claim 1 in which the framework is composed of a biodegradable material.
 5. The three-dimensional cell culture of claim 4 in which the biodegradable material is cotton, polyglycolic acid, cat gut sutures, cellulose, gelatin, or dextran.
 6. The three-dimensional cell culture of claim 1 in which the framework is composed of a non-biodegradable material.
 7. The three-dimensional cell culture of claim 6 in which the non-biodegradable material is a polyamide, a polyester, a polystyrene, a polypropylene, a polyacrylate, a polyvinyl, a polycarbonate, a polytetrafluoroethylene, or a nitrocellulose compound.
 8. The three-dimensional cell culture of claim 4, 5, 6 or 7 in which the framework is pre-coated with collagen.
 9. The three-dimensional cell culture of claim 8 in which the framework is a mesh.
 10. The three-dimensional cell culture of claim 1, 2, 3, 4, 5, 6 or 7 in which the framework is a mesh.
 11. A method for culturing pancreatic cells in vitro, comprising culturing pancreatic acinar cells inoculated onto a living stromal tissue prepared in vitro, said living stromal tissue comprising stromal cells and connective tissue proteins naturally secreted by the stromal cells attached to and substantially enveloping a framework composed of a biocompatible, non-living material formed into a three-dimensional structure having interstitial spaces bridged by the stromal cells.
 12. The method according to claim 11 in which the stromal cells are fibroblasts.
 13. The method according to claim 11 in which the stromal cells are a combination of fibroblasts and endothelial cells, pericytes, macrophages, monocytes, leukocytes, plasma cells, mast cells or adipocytes.
 14. The method according to claim 11 in which the framework is composed of a biodegradable material.
 15. The method according to claim 14 in which the biodegradable material is cotton, polyglycolic acid, cat gut sutures, cellulose, gelatin, or dextran.
 16. The method according to claim 11 in which the framework is composed of a non-biodegradable material.
 17. The method according to claim 16 in which the non-biodegradable material is a polyamide, a polyester, a polystyrene, a polypropylene, a polyacrylate, a polyvinyl, a polycarbonate, a polytetrafluoroethylene, or a nitrocellulose compound.
 18. The method according to claim 14, 15, 16 or 17 in which the framework is pre-coated with collagen.
 19. The method according to claim 18 in which the framework is a mesh.
 20. The method according to claim 11, 12, 13, 14, 15, 16 or 17 in which the framework is a mesh.
 21. A method for transplantation or implantation of pancreatic cells in vivo comprising implanting in vivo the pancreatic acinar cells cultured on a living stromal tissue prepared in vitro, said living stromal tissue comprising stromal cells and connective tissue proteins naturally secreted by the stromal cells attached to and substantially enveloping a framework composed of a biocompatible, non-living material formed into a three-dimensional structure having interstitial spaces bridged by the stromal cells so that a tissue equivalent is formed.
 22. The method according to claim 21 in which the stromal cells are fibroblasts.
 23. The method according to claim 21 in which the stromal cells are a combination of fibroblasts and endothelial cells, pericytes, macrophages, monocytes, leukocytes, plasma cells, mast cells or adipocytes.
 24. The method according to claim 21 in which the framework is composed of a biodegradable material.
 25. The method according to claim 24 in which the biodegradable material is cotton, polyglycolic acid, cat gut sutures, cellulose, gelatin, or dextran.
 26. The method according to claim 21 in which the framework is composed of a non-biodegradable material.
 27. The method according to claim 26 in which the non-biodegradable material is a polyamide, a polyester, a polystyrene, a polypropylene, a polyacrylate, a polyvinyl, a polycarbonate, a polytetrafluoroethylene, or a nitrocellulose compound.
 28. The method according to claim 24, 25, 26 or 27 in which the framework is pre-coated with collagen.
 29. The method according to claim 28 in which the framework is a mesh.
 30. The method according to claim 21, 22, 23, 24, 25, 26 or 27 in which the framework is a mesh.
 31. A method for determining the effect of a drug on pancreatic acinar cells in culture comprising:(a) exposing a three-dimensional pancreatic cell culture to the drug, in which the three-dimensional cell culture comprises pancreatic acinar cells grown on a living stromal tissue prepared in vitro, said living stromal tissue comprising stromal cells and connective tissue proteins naturally secreted by the stromal cells attached to and substantially enveloping a framework composed of a biocompatible, non-living material formed into a three-dimensional structure having interstitial spaces bridged by the stromal cells; and (b) determining the effect of the drug on the pancreatic acinar cells in culture.
 32. The method for determining the effect of a drug according to claim 31 in which the stromal cells are fibroblasts.
 33. The method for determining the effect of a drug according to claim 31 in which the stromal cells are a combination of fibroblasts and endothelial cells, pericytes, macrophages, monocytes, leukocytes, plasma cells, mast cells or adipocytes.
 34. The method for determining the effect of a drug according to claim 31 in which the framework is composed of a biodegradable material.
 35. The method for determining the effect of a drug according to claim 34 in which the biodegradable material is cotton, polyglycolic acid, cat gut sutures, cellulose, gelatin, or dextran.
 36. The method for determining the effect of a drug according to claim 31 in which the framework is composed of a non-biodegradable material.
 37. The method for determining the effect of a drug according to claim 36 in which the non-biodegradable material is a polyamide, a polyester, a polystyrene, a polypropylene, a polyacrylate, a polyvinyl, a polycarbonate, a polytetrafluoroethylene, or a nitrocellulose compound.
 38. The method for determining the effect of a drug according to claim 34, 35, 36 or 37 in which the framework is pre-coated with collagen.
 39. The method according to claim 38 in which the framework is a mesh.
 40. The method according to claim 31, 32, 33, 34, 35, 36 or 37 in which the framework is a mesh.
 41. A method for studying the mechanisms of a disease or condition in a patient, which disease or condition has a detectable effect on pancreatic acinar cells in culture, comprising:(a) culturing pancreatic acinar cells from a sample obtained from the patient onto a living stromal tissue prepared in vitro, said living stromal tissue comprising stromal cells and connective tissue proteins naturally secreted by the stromal cells attached to and substantially enveloping a framework composed of a biocompatible, non-living material formed into a three-dimensional structure having interstitial spaces bridged by the stromal cells; (b) culturing the inoculated living stromal tissue in a nutrient medium so that the inoculated pancreatic acinar cells proliferate in culture; and (c) analyzing the proliferated pancreatic acinar cells in culture for markers of the disease or condition.
 42. The method according to claim 41 in which the stromal cells are fibroblasts.
 43. The method according to claim 41 in which the stromal cells are a combination of fibroblasts and endothelial cells, pericytes, macrophages, monocytes, leukocytes, plasma cells, mast cells or adipocytes.
 44. The method according to claim 41 in which the framework is composed of a biodegradable material.
 45. The method according to claim 44 in which the biodegradable material is cotton, polyglycolic acid, cat gut sutures, cellulose, gelatin, or dextran.
 46. The method according to claim 41 in which the framework is composed of a non-biodegradable material.
 47. The method according to claim 46 in which the non-biodegradable material is a polyamide, a polyester, a polystyrene, a polypropylene, a polyacrylate, a polyvinyl, a polycarbonate, a polytetrafluoroethylene, or a nitrocellulose compound.
 48. The method according to claim 44, 45, 46 or 47 in which the framework is pre-coated with collagen.
 49. The method according to claim 41, 42, 43, 44, 45, 46 or 47 in which the framework is a mesh.
 50. The method according to claim 48 in which the framework is a mesh. 